US10392378B2

US10392378B2 - Derivatives of 5-phenyl- or 5-heteroarylathiazol-2-carboxylic amide useful for the treatment of inter alia cystic fibrosis

Info

Publication number: US10392378B2
Application number: US15/539,397
Authority: US
Inventors: Cecilia M. Bastos; Benito Munoz; Bradley Tait
Original assignee: Proteostasis Therapeutics Inc
Current assignee: Proteostasis Therapeutics Inc
Priority date: 2014-12-23
Filing date: 2015-12-23
Publication date: 2019-08-27
Anticipated expiration: 2035-12-23
Also published as: US20170369482A1; WO2016105477A1; CA2971850A1

Abstract

The present disclosure is based, in part, on the discovery that disclosed compounds such as those having Formula (IIIa), (III), or (IV) can increase cystic fibrosis transmembrane conductance regulator (CFTR) activity as measured in human bronchial epithelial (hBE) cells.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of PCT/US2015/000202, filed Dec. 23, 2015, which claims the benefit of, and priority to, U.S. provisional application Ser. Nos. 62/102,208, filed Jan. 12, 2015, and 62/096,416, filed Dec. 23, 2014, the contents of each of which are hereby incorporated by reference herein in their entirety.

BACKGROUND

Cells normally maintain a balance between protein synthesis, folding, trafficking, aggregation, and degradation, referred to as protein homeostasis, utilizing sensors and networks of pathways (Sitia et al., Nature 426: 891-894, 2003; Ron et al., Nat Rev Mol Cell Biol 8: 519-529, 2007). The cellular maintenance of protein homeostasis, or proteostasis, refers to controlling the conformation, binding interactions, location and concentration of individual proteins making up the proteome. Protein folding in vivo is accomplished through interactions between the folding polypeptide chain and macromolecular cellular components, including multiple classes of chaperones and folding enzymes, which minimize aggregation (Wiseman et al., Cell 131: 809-821, 2007). Whether a given protein folds in a certain cell type depends on the distribution, concentration, and subcellular localization of chaperones, folding enzymes, metabolites and the like (Wiseman et al.). Cystic fibrosis and other maladies of protein misfolding arise as a result of an imbalance in the capacity of the protein homeostasis (proteostasis) environment to handle the reduced energetic stability of misfolded, mutated proteins that are critical for normal physiology (Balch et al., Science 319, 916-9 (2008); Powers, et al., Annu Rev Biochem 78, 959-91 (2009); Hutt et al., FEBS Lett 583, 2639-46 (2009)).

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes a multi-membrane spanning epithelial chloride channel (Riordan et al., Annu Rev Biochem 77, 701-26 (2008)). Approximately ninety percent of patients have a deletion of phenylalanine (Phe) 508 (ΔF508) on at least one allele. This mutation results in disruption of the energetics of the protein fold leading to degradation of CFTR in the endoplasmic reticulum (ER). The ΔF508 mutation is thus associated with defective folding and trafficking, as well as enhanced degradation of the mutant CFTR protein (Qu et al., J Biol Chem 272, 15739-44 (1997)). The loss of a functional CFTR channel at the plasma membrane disrupts ionic homeostasis (Cl⁻, Na⁺, HCO₃ ⁻) and airway surface hydration leading to reduced lung function (Riordan et al.). Reduced periciliary liquid volume and increased mucus viscosity impede mucociliary clearance resulting in chronic infection and inflammation, phenotypic hallmarks of CF disease (Boucher, J Intern Med 261, 5-16 (2007)). In addition to respiratory dysfunction, ΔF508 CFTR also impacts the normal function of additional organs (pancreas, intestine, gall bladder), suggesting that the loss-of-function impacts multiple downstream pathways that will require correction.

In addition to cystic fibrosis, mutations in the CFTR gene and/or the activity of the CFTR channel has also been implicated in other conditions, including for example, congenital bilateral absence of vas deferens (CBAVD), acute, recurrent, or chronic pancreatitis, disseminated bronchiectasis, asthma, allergic pulmonary aspergillosis, smoking-related lung diseases, such as chronic obstructive pulmonary disease (COPD), dry eye disease, Sjogren's syndrome and chronic sinusitis, (Sloane et al. (2012), PLoS ONE 7(6): e39809.doi:10.1371/journal. pone.0039809; Bombieri et al. (2011), J Cyst Fibros. 2011 June; 10 Suppl 2:S86-102; (Albert et al. (2008). Clinical Respiratory Medicine, Third Ed., Mosby Inc.; Levin et al. (2005), Invest Ophthalmol Vis Sci., 46(4):1428-34; Froussard (2007), Pancreas 35(1): 94-5).

There remains a need in the art for compounds, compositions and methods of increasing CFTR activity as well as for methods of treating CF, other CFTR-related diseases, and other maladies of protein misfolding.

SUMMARY

The present disclosure is based, in part, on the discovery that disclosed compounds can increase cystic fibrosis transmembrane conductance regulator (CFTR) activity as measured in human bronchial epithelial (hBE) cells.

For example, disclosed herein are compounds having the Formula (III) or (IV):

and pharmaceutically acceptable salts, stereoisomers, and prodrugs thereof, wherein:

X₃is selected from the group consisting of O, S, and NR_hh;

pp is 1, 2, or 3;

R₁₁is independently selected for each occurrence from the group consisting of hydrogen, halogen, C_1-4alkyl (optionally substituted by one, two or three halogens);

L₁is selected from the group consisting of C_1-6alkylene, C_3-6cycloalkylene, C_3-6cycloalkylene-C_1-4alkylene, C_1-3alkylene-NR_hh—S(O)_w—, —C_1-3alkylene-S(O)_w—NR_hh—, C_3-6cycloalkylene-C_0-2alkylene-S(O)_w—NR_hh, and C_3-6cycloalkylene-C_0-2alkylene NR_hh—S(O)_w—, wherein L₁may be optionally substituted by one, two or three substituents selected from the group consisting of halogen, hydroxyl, C_1-3alkyl (optionally substituted by one, two or three substituents each selected independently from R_ff);

R₄₄is selected from the group consisting of H, halogen, hydroxyl, C_1-3alkoxy, heterocycle, and a 5-6 membered monocyclic or 8-10 membered bicyclic heteroaryl having one, two or three heteroatoms each selected from O, N, and S; wherein the heterocycle and the heteroaryl may be optionally substituted by one or two substituents each selected independently from R_gg;

R_ffis selected for each occurrence from group consisting of halogen, hydroxyl, C_1-4alkyl, C_1-4alkyoxy, C_2-4alkenyl, C_3-6cycloalkyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)_w—C_1-3alkyl, where w is 0, 1, or 2, wherein C_1-4alkyl, C_1-4alkyoxy, C_2-4alkenyl and C_3-6cycloalkyl may be optionally substituted by one, two or three substituents each independently selected from the group consisting of halogen, hydroxyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)_w—C_1-3alkyl;

R_ggis selected for each occurrence from group consisting of halogen, hydroxyl, C_1-6alkyl, C_1-6alkyoxy, C_2-6alkenyl, C_3-6cycloalkyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)_w—C_1-3alkyl, where w is 0, 1, or 2, wherein C_1-6alkyl, C_1-6alkyoxy, C_2-6alkenyl and C_3-6cycloalkyl may each be optionally substituted by one, two or three substituents each independently selected from the group consisting of halogen, C_1-6alkyl, C_1-6alkoxy, hydroxyl, C(O)OH, —C(O)OC_1-6alkyl, —O—C_3-6cycloalkyl, —O-heterocycle, —O-heteroaryl, —O-phenyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)w-C_1-3alkyl;

w is 0, 1 or 2; and

R_hhis selected for each occurrence from the group consisting of H, C_1-6alkyl and C_3-6cycloalkyl.

Also contemplated herein are pharmaceutical compositions that include a disclosed compound such as those compounds having Formula (III) or (IV) and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions can include at least one additional CFTR modulator as described anywhere herein or at least two additional CFTR modulators, each independently as described anywhere herein.

In additional embodiments, a method of enhancing (e.g., increasing) cystic fibrosis transmembrane conductance regulator (CFTR) activity in a subject in need thereof is provided comprising administering to said subject an effective amount of a compound of Formula (III) or (IV).

In certain of these embodiments, the activity of one or more (e.g., one or two) mutant CFTRs (e.g., ΔF508, S549N, G542X, G551D, R117H, N1303K, W1282X, R553X, 621+1G>T, 1717-1G>A, 3849+10kbC>T, 2789+5G>A, 3120+1G>A, I507del, R1162X, 1898+1G>A, 3659delC, G85E, D1152H, R560T, R347P, 2184insA, A455E, R334W, Q493X, and 2184delA CFTR) is enhanced (e.g., increased). In certain embodiments, ΔF508 CFTR activity is enhanced (e.g., increased). In other embodiments, the activities of two mutant CFTRs (e.g., ΔF508 and G551D; ΔF508 and A455E; or G542X; Δ508F) are enhanced (e.g., increased).

In certain of these embodiments, the subject (e.g., a human patient) is suffering from a disease associated with decreased CFTR activity (e.g., cystic fibrosis, congenital bilateral absence of vas deferens (CBAVD), acute, recurrent, or chronic pancreatitis, disseminated bronchiectasis, asthma, allergic pulmonary aspergillosis, chronic obstructive pulmonary disease (COPD), chronic sinusitis, dry eye disease, protein C deficiency, A-β-lipoproteinemia, lysosomal storage disease, type 1 chylomicronemia, mild pulmonary disease, lipid processing deficiencies, type 1 hereditary angioedema, coagulation-fibrinolyis, hereditary hemochromatosis, CFTR-related metabolic syndrome, chronic bronchitis, constipation, pancreatic insufficiency, hereditary emphysema, Sjogren's syndrome, familial hypercholesterolemia, I-cell disease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism, myleoperoxidase deficiency, primary hypoparathyroidism, melanoma, glycanosis CDG type 1, congenital hyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, nephrogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy, Pick's disease, Huntington's disease, spinocerebellar ataxia type I, spinal and bulbar muscular atrophy, dentatorubral pallidoluysian, myotonic dystrophy, hereditary Creutzfeldt-Jakob disease (due to prion protein processing defect), Fabry disease, and Straussler-Scheinker syndrome). In certain embodiments, the disease is cystic fibrosis.

In yet additional aspects, the disclosure is directed to treating a patient suffering from cystic fibrosis comprising administering to said patient an effective amount of a disclosed compound.

In some embodiments, the methods described herein can further include administering an additional CFTR modulator or administering at least two additional CFTR modulators. In certain embodiments, at least one CFTR modulator is a CFTR corrector (e.g., VX-809, VX-661, VX-983, VX-983, VX-152, VX-440, GLPG2222 and GLPG2665) or potentiator (e.g., ivacaftor and genistein). In certain of these embodiments, one of the at least two additional therapeutic agents is a CFTR corrector (e.g., VX-809, VX-661, VX-983, VX-152, VX-440, GLPG2222 and GLPG2665) and the other is a CFTR potentiator (e.g., ivacaftor and genistein).

In a further aspect, a method of identifying a candidate agent that increases CFTR activity is provided, which includes: (i) contacting a cell that expresses a CFTR protein with the candidate agent and a disclosed compound; (ii) measuring the CFTR activity in the cell in the presence of the candidate agent and the disclosed compound; and (iii) comparing the CFTR activity to that in the absence of the test agent, wherein an increase in CFTR activity in the presence of the test agent indicates that the agent increases CFTR activity. In certain embodiments, the cell expresses a mutant CFTR protein. In certain embodiments, CFTR activity is measured by measuring chloride channel activity of the CFTR, and/or other ion transport activity. In certain of these embodiments, the method is high-throughput. In certain of these embodiments, the candidate agent is a CFTR corrector or a CFTR potentiator.

DETAILED DESCRIPTION

As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. For example, the term “an agent” encompasses both a single agent and a combination of two or more agents.

As discussed above, the present disclosure is directed in part to compounds as described herein, or a pharmaceutically acceptable salt, prodrug or solvate thereof, and pharmaceutical compositions, methods of increasing CFTR activity and methods of treating cystic fibrosis.

For example, disclosed herein are compounds having the Formula (III) or (IV):

X₃is selected from the group consisting of O, S, and NR_hh;

pp is 1, 2, or 3;

w is 0, 1 or 2; and

In an exemplary embodiment, L₁is C_1-3alkylene or C_3-5cycloalkylene.

For example, provided herein is a disclosed compound with the formula:

wherein qq is 0 or 1, and pharmaceutically acceptable salts thereof (where R₄₄, R₁₁, and pp are provided herein).

For example, a disclosed compound may include those having the structures:

and pharmaceutically acceptable salts thereof.

In certain embodiments, R₄₄as provided above, may be selected from the group consisting of: pyrrolidinyl, piperidinyl, tetrahydropyranyl, and tetrahydrofuranyl. In other embodiments, R₄₄is selected from the group consisting of:

wherein X independently for each occurrence is selected from the group consisting of O, S, NR_hh, C, C(R₈₈), and C(R₈₈)(R₉₉); X₂independently for each occurrence is selected from the group consisting of O, S and NR_hh; R″ is H or C_1-4alkyl, each R₆₆, R₇₇, R₈₈and R₉₉is independently selected for each occurrence from H and R_gg, and n is 0, 1, 2, or 3.

Each of R₆₆, R₇₇, R₈₈and R₉₉, in certain embodiments, is independently selected for each occurrence from the group consisting of hydrogen, halogen, hydroxyl, C_1-6alkyl, C_3-6cycloalkyl, and heterocycle, wherein C_1-6alkyl, C_3-6cycloalkyl, and heterocycle are optionally substituted by one, two or three substituents each independently selected from the group consisting of hydroxyl, C_1-6alkyl, C_1-6alkoxy (optionally substituted by C_3-6cycloalkyl, heterocycle, —C_1-2alkyl-heterocycle and C_1-2alkyl-C_3-6cycloalkyl), —S(O)_w—C_1-3alkyl (w is 0, 1, or 2) and —NR′S(O)₂C_1-6alkyl; and R′ is independently selected for each occurrence from H and C_1-4alkyl. As provided herein, pp may be 0, 1 or 2, and/or R₁₁is selected from H, F, or methyl.

Also disclosed herein are compounds such as those having the Formula (IIIa):

or a pharmaceutically acceptable salt, prodrug or solvate thereof, wherein:

R₁is selected from the group consisting of:

R₂is selected from the group consisting of optionally substituted aryl and optionally substituted heteroaryl;

R_3ais selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted aryl, halo, OR_c, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)_nR_c, N(R_d)(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nNR_dR_d, NR_dS(O)_nR_c, S(O)_nR_c, S(O)_nNR_dR_d, OC(O)OR_c, (C═NR_d)R_c, optionally substituted heterocyclic and optionally substituted heteroaryl;

R_4ais selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted aryl, halo, OR_c, S(O)_nR_c, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)R_c, N(R_d)(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nR_dR_d, NR_dS(O)_nR_c, S(O)NR_dR_d, OC(O)OR_c, (C═NR_d)R_c, optionally substituted heterocyclic and optionally substituted heteroaryl;

R_4bis selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted aryl, optionally substituted heterocyclic and optionally substituted heteroaryl;

R_ais selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, C(O)OR_c, C(O)R_c, C(O)C(O)R_cand S(O)_nR_c;

or alternatively, R_aand the nitrogen atom to which it is attached is taken together with an adjacent C(R_b1)(R_b1) or C(R_b2)(R_b2) to form an optionally substituted, 4- to 12-membered heterocyclic ring containing one or more ring nitrogen atoms, wherein said heterocyclic ring optionally contains one or more ring heteroatoms selected from oxygen and sulfur;

each R_b1and R_b2is independently selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted heterocyclic, optionally substituted aryl, optionally substituted heteroaryl, halo, OR_c, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)_nR_c, N(R_d)(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nNR_dR_d, NR_dS(O)_nR_c, S(O)_nR_c, S(O)_nNR_dR_d, OC(O)OR_cand (C═NR_d)R_c; or alternatively, two geminal R_b1groups or two geminal R_b2groups and the carbon to which they are attached are taken together to form a C(O) group, or yet alternatively, two geminal R_b1groups or two geminal R_b2groups are taken together with the carbon atom to which they are attached to form a spiro C₃-C₁₂cycloalkyl, a spiro C₃-C₁₂cycloalkenyl, a spiro heterocyclic, a spiro aryl or spiro heteroaryl, each optionally substituted;

Y is selected from the group consisting of S(O)_n, NR_d, NR_dS(O)_n, NR_dS(O)_nNR_d, NR_dC(O), NR_dC(O)O, NR_dC(O)C(O), NR_dC(O)NR_d, S(O)_nNR_d, and O;

each R_cis independently selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted heterocyclic, optionally substituted aryl and optionally substituted heteroaryl;

each R_dis independently selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₁-C₁₀alkoxy, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted heterocyclic, optionally substituted aryl and optionally substituted heteroaryl; or two geminal R_dgroups are taken together with the nitrogen atom to which they are attached to form an optionally substituted heterocyclic or an optionally substituted heteroaryl;

k is 0 or 1;

m is 0, 1, 2, 3, 4, or 5;

each n is independently 0, 1 or 2.

In some embodiments, m is 0, 1 or 2 (e.g., m is 1). In some embodiments, k is 0. In some embodiments, m is 0, 1 or 2, and k is 0. In other embodiments, m is 1, and k is 0.

In some embodiments, R_3ais hydrogen.

In some embodiments, R_ais hydrogen or C₁-C₄alkyl (optionally substituted by 1, 2 or 3 halogens).

In some embodiments, each of R_b1and R_b2is independently selected from hydrogen, hydroxyl, C_1-4alkoxy (optionally substituted by one, two or three substituents independently selected from halogen and hydroxyl) and C₁-C₄alkyl (optionally substituted by one, two or three substituents independently selected from halogen, hydroxyl, and C_1-4alkoxy). In certain embodiments, R_b1and R_b2for each occurrence are hydrogen.

In some embodiments, R₂is selected from the group consisting of phenyl and a 5-6 membered heteroaryl having one or two heteroatoms each selected from N, S, and O, wherein R₂is optionally substituted by one or two substituents each independently selected from the group consisting of halogen and C₁-C₄alkyl (optionally substituted by one, two or three halogens).

In certain embodiments, R₂is phenyl. In other embodiments, R₂is phenyl is substituted with one or two R₅, wherein each R₅is independently selected from the group consisting of optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, and halo.

In other embodiments, R₂is selected from the group consisting of optionally substituted thienyl, optionally substituted furanyl and optionally substituted pyridinyl.

In some embodiments, R_4ais selected from the group consisting of optionally substituted C₁-C₆alkyl, optionally substituted C₃-C₇cycloalkyl, phenyl, OR_c, C(O)OR_c, C(O)R_c, optionally substituted heterocycle and optionally substituted heteroaryl, wherein R_cis selected, independently for each occurrence, from the group consisting of H and C_1-6alkyl.

In certain embodiments R_4ais a heterocycle, or a 5-6 membered monocyclic or a 8-10 membered bicyclic heteroaryl having one, two or three heteroatoms selected from N, S or O, wherein the heterocycle or heteroaryl are optionally substituted by one, two or three substituents independently selected for each occurrence from the group consisting of halogen, C_1-6alkyl (optionally substituted by one, two or three substituents each independently selected from halogen and hydroxyl), C_1-6alkoxy (optionally substituted by one, two or three halogens), hydroxyl, and NR_dR_dwherein R_dis independently for each occurrence selected from H and C_1-4alkyl, or the two R_ds taken together with the N to which they are attached form a heterocyclic ring. For example, R_4acan be selected from the group consisting of tetrahydropyranyl, thiadiazolyl, tetrahydrofuranyl, and morpholinyl. As another example, R_4acan be a monocyclic heteroaryl containing one, two or three ring nitrogen atoms. As a further example, R_4acan be selected from the group consisting of furanyl, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, isoxazolyl, triazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, thienyl, piperazinyl, and benzimidazolyl, each optionally substituted.

In certain embodiments, R_4ais selected from the group consisting of:

wherein each X is independently O, S or NR_g;

each R_gis independently selected from the group consisting of hydrogen, C₁-C₄alkyl, C₃-C₆cycloalkyl; and

each R₆, R₇and R₈is independently selected for each occurrence from the group consisting of hydrogen, C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₁₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloalkenyl, phenyl, heterocycle, heteroaryl, halo, hydroxyl, carboxyl, OR_c, NR_dR_d, C(O)OR_c, CN, C(O)R_c, wherein the C_1-6alkyl, C₂-C₆alkenyl, C₂-C₁₆alkynyl, C₃-C₇cycloalkyl, C₃-C₇cycloalkenyl, phenyl, heterocycle, and heteroaryl of R₆, R₇and R₈may each be optionally substituted by one, two or three substituents selected from halo, hydroxyl, C_1-6alkyl and C_1-6alkoxy;

R_cis C_1-4alkyl; and

R_dis independently for each occurrence selected from the group consisting of H and C_1-4alkyl, or the two R_ds taken together with the N to which they are attached form a heterocyclic ring.

In certain embodiments, R_4ais an optionally substituted C₃-C₇cycloalkyl (e.g., optionally substituted cyclopropyl or an optionally substituted cyclobutyl).

In certain of these embodiments, R_4ais substituted with a substituent having the formula:

wherein each R_his independently selected for each occurrence from the group consisting of hydrogen, halo, hydroxyl, C₁-C₆alkyl, and C₃-C₆cycloalkyl, or two geminal R_hgroups are independently taken together with the carbon atom to which they are attached to form an optionally substituted carbocyclic or heterocycle;

R₉is selected from the group consisting of hydrogen, halo, CN, hydroxyl, methyl (optionally substituted by one, two or three substituents selected from halogen and hydroxyl), C₂-C₄alkenyl, C₂-C₄alkynyl, C₃-C₆cycloalkyl, C_1-6alkoxy, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)_nR_c, NR_d(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nNR_dR_d, NR_dS(O)_nR_c, S(O)_nR_c, S(O)_nNR_dR_d, OC(O)OR_c, (C═NR_d)R_c;

R_cis independently selected for each occurrence from the group consisting of H, C₁-C₆alkyl, C_3-6cycloalkyl, heterocycle, and heteroaryl;

R_dis independently selected for each occurrence from H and C_1-4alkyl, or the two R_ds taken together with the N to which they are attached form a heterocyclic ring; and p is 0, 1, or 2.

For example, R_4acan be selected from the group consisting of:

wherein
each R₁₀is independently selected from the group consisting of hydrogen, optionally substituted C₁-C₆alkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆alkynyl, optionally substituted C₃-C₆cycloalkyl, optionally substituted C₃-C₆cycloalkenyl, optionally substituted aryl, halo, OR_c, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)_nR_c, NR_d(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nNR_dR_d, NR_dS(O)_nR_c, S(O)_nR_c, S(O)_nNR_dR_d, OC(O)OR_c, (C═NR_d)R_c, optionally substituted heterocyclic and optionally substituted heteroaryl; alternatively, two geminal R₁₀groups are taken together with the carbon atom to which they are attached to form a spiro C₃-C₇cycloalkyl, a spiro C₃-C₇cycloalkenyl, a spiro heterocyclic, a spiro aryl or spiro heteroaryl, each optionally substituted; or yet alternatively, two vicinal R₁₀groups are taken together with the carbon atoms to which they are attached to form a fused, optionally substituted cyclic group selected from the group consisting of C₄-C₈cycloalkyl, C₄-C₈cycloalkenyl, 4- to 8-membered heterocyclic, aryl and heteroaryl, each optionally substituted; or further alternatively, two R₁₀groups attached to non-adjacent carbon atoms are taken together with the carbon atoms to which they are attached to form a bridged cyclic group selected from the group consisting of C₃-C₈cycloalkyl, C₃-C₈cycloalkenyl, and 4- to 8-membered heterocyclic, each optionally substituted;

- each R_his independently selected from the group consisting of hydrogen, halo, optionally substituted C₁-C₁₀alkyl, and optionally substituted C₃-C₆cycloalkyl, or two geminal R_bgroups are independently taken together with the carbon atom to which they are attached to form an optionally substituted heterocyclic or an optionally substituted heteroaryl;
- R₉is selected from the group consisting of hydrogen, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted aryl, halo, OR_c, NR_dR_d, C(O)OR_c, NO₂, CN, C(O)R_c, C(O)C(O)R_c, C(O)NR_dR_d, NR_dC(O)R_c, NR_dS(O)_nR_c, NR_d(COOR_c), NR_dC(O)C(O)R_c, NR_dC(O)NR_dR_d, NR_dS(O)_nNR_dR_d, NR_dS(O)_nR_c, S(O)_nR_c, S(O)_nNR_dR_d, OC(O)OR_c, (C═NR_d)R_c, optionally substituted heterocyclic and optionally substituted heteroaryl; and p is 0, 1, or 2.

In some embodiments, Y is S, S(O)₂or S(O)₂NR_d.

In some embodiments, R_4bis heterocycle or a 5-6 membered monocyclic or a 8-10 membered bicyclic heteroaryl having one, two or three heteroatoms selected from N, S or O, wherein the heterocycle or heteroaryl are optionally substituted by one, two or three substituents independently selected for each occurrence from the group consisting of halogen, C_1-6alkyl (optionally substituted by one, two or three substituents each independently selected from halogen and hydroxyl), C_1-6alkoxy (optionally substituted by one, two or three halogens), hydroxyl, and NR_dR_dwherein R_dis independently for each occurrence selected from H and C_1-4alkyl, or the two R_ds taken together with the N to which they are attached form a heterocyclic ring). For example, R_4bcan be selected from the group consisting of furanyl, pyridinyl, pyrazinyl, pyrazolyl, imidazolyl, isoxazolyl, triazolyl, thiazolyl, oxadiazolyl, thiadiazolyl, thienyl, piperazinyl, and benzimidazolyl, each optionally substituted.

Exemplary compounds are shown below in Table 1:

TABLE 1

#	Structure

1

2

3

4

5

6

7

8

9

10

11

12

Also contemplated herein are pharmaceutical compositions that include a disclosed compound such as those compounds having Formula (IIIa), (III), or (IV) and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions can include at least one additional CFTR modulator as described anywhere herein or at least two additional CFTR modulators, each independently as described anywhere herein.

It is to be understood that the specific embodiments described herein can be taken in combination with other specific embodiments delineated herein. For example, as discussed above, in some embodiments, R_2ais fluoro, and in some embodiments described above, A is an optionally substituted imidazolyl or pyrazolyl. The disclosure thus contemplates for example, a compound of Formula (IIIa), (III), or (IV), wherein R_2ais fluoro and A is an optionally substituted imidazolyl or pyrazolyl.

The features and other details of the disclosure will now be more particularly described. Before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

It will be appreciated that the description of the present invention herein should be construed in congruity with the laws and principals of chemical bonding.

The term “alkyl”, as used herein, unless otherwise indicated, refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; for example, “C₁-C₁₀alkyl” denotes alkyl having 1 to 10 carbon atoms, and straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as C_1-6alkyl, C_1-4alkyl, and C_1-3alkyl, respectively. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, 2-methylbutyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl.

The term, “alkenyl”, as used herein, refers to both straight and branched-chain moieties having the specified number of carbon atoms and having at least one carbon-carbon double bond. Exemplary alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C_2-6alkenyl, and C_3-4alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.

The term, “alkynyl”, as used herein, refers to both straight and branched-chain moieties having the specified number or carbon atoms and having at least one carbon-carbon triple bond.

The term “cycloalkyl,” as used herein, refers to saturated cyclic alkyl moieties having 3 or more carbon atoms, for example, 3-10, 3-6, or 4-6 carbons, referred to herein as C_3-10cycloalkyl, C_3-6cycloalkyl or C_4-6cycloalkyl, respectively for example, Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and adamantyl.

The term “cycloalkenyl,” as used herein, refers to cyclic alkenyl moieties having 3 or more carbon atoms.

The term “cycloalkynyl,” as used herein, refers to cyclic alkynyl moieties having 5 or more carbon atoms.

“Alkylene” means a straight or branched, saturated aliphatic divalent radical having the number of carbons indicated. “Cycloalkylene” refers to a divalent radical of carbocyclic saturated hydrocarbon group having the number of carbons indicated.

The term “alkoxy” as used herein refers to a straight or branched alkyl group attached to oxygen (alkyl-O—). Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as C_1-6alkoxy, and C_2-6alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

The term “heterocyclic” or “heterocycle” encompasses heterocycloalkyl, heterocycloalkenyl, heterobicycloalkyl, heterobicycloalkenyl, heteropolycycloalkyl, heteropolycycloalkenyl, and the like unless indicated otherwise. Heterocycloalkyl refers to cycloalkyl groups containing one or more heteroatoms (O, S, or N) within the ring. Heterocycloalkenyl as used herein refers to cycloalkenyl groups containing one or more heteroatoms (O, S or N) within the ring. Heterobicycloalkyl refers to bicycloalkyl groups containing one or more heteroatoms (O, S or N) within a ring. Heterobicycloalkenyl as used herein refers to bicycloalkenyl groups containing one or more heteroatoms (O, S or N) within a ring. a heterocycle can refer to, for example, a saturated or partially unsaturated 4- to 12 or 4-10-membered ring structure, including bridged or fused rings, and whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran or dihydrofuran, etc.

Cycloalkyl, cycloalkenyl, and heterocyclic groups also include groups similar to those described above for each of these respective categories, but which are substituted with one or more oxo moieties.

The term “aryl”, as used herein, refers to mono- or polycyclic aromatic carbocyclic ring systems. A polycyclic aryl is a polycyclic ring system that comprises at least one aromatic ring. Polycyclic aryls can comprise fused rings, covalently attached rings or a combination thereof. The term “aryl” embraces aromatic radicals, such as, phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. An aryl group may be substituted or unsubstituted. In some embodiments, the aryl is a C₄-C₁₀aryl. Examples of optionally substituted aryl are phenyl, substituted phenyl, naphthyl and substituted naphthyl.

The term “heteroaryl”, as used herein, refers to aromatic carbocyclic groups containing one or more heteroatoms (O, S, or N) within a ring. A heteroaryl group, unless indicated otherwise, can be monocyclic or polycyclic. A heteroaryl group may additionally be substituted or unsubstituted. Heteroaryl groups can also include ring systems substituted with one or more oxo moieties. A polycyclic heteroaryl can comprise fused rings, covalently attached rings or a combination thereof. A polycyclic heteroaryl is a polycyclic ring system that comprises at least one aromatic ring containing one or more heteroatoms within a ring. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, triazinyl, isoindolyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl, tetrahydroisoquinolyl, benzofuryl, furopyridinyl, pyrolopyrimidinyl, thiazolopyridinyl, oxazolopyridinyl and azaindolyl. The foregoing heteroaryl groups may be C-attached or heteroatom-attached (where such is possible). For instance, a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). In some embodiments, the heteroaryl is 4- to 12-membered heteroaryl. In yet other embodiments, the heteroaryl is a mono or bicyclic 4- to 10-membered heteroaryl.

The term “substituted” refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, and unless indicated otherwise, —C₁-C₁₂alkyl, —C₂-C₁₂alkenyl, —C₂-C₁₂alkynyl, —C₃-C₁₂cycloalkyl, —C₃-C₁₂cycloalkenyl, C₃-C₁₂cycloalkynyl, -heterocyclic, —F, —Cl, —Br, —I, —OH, —NO₂, —N₃, —CN, —NH₂, oxo, thioxo, —NHR_x, —NR_xR_x, dialkylamino, -diarylamino, -diheteroarylamino, —OR_x, —C(O)R_y, —C(O)C(O)R_y, —OCO₂R_y, —OC(O)R_y, OC(O)C(O)R_y, —NHC(O)R_y, —NHCO₂R_y, —NHC(O)C(O)R_y, NHC(S)NH₂, —NHC(S)NHR_x, —NHC(NH)NH₂, —NHC(NH)NHR_x, —NHC(NH)R_x, —C(NH)NHR_x, and (C═NR_x)R_x; —NRxC(O)R_x, —NR_xC(O)N(R_x)₂, —NRxCO₂R_y, —NRxC(O)C(O)R_y, —NR_xC(S)NH₂, —NR_xC(S)NHR_x, —NR_xC(NH)NH₂, —NR_xC(NH)NHR_x, —NR_xC(NH)R_x, —C(NRx)NHR_x—S(O)R_y, —NHSO₂R_x, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl, -polyalkoxyalkyl, -polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH, —S—R_x, or -methylthiomethyl, wherein R_xis selected from the group consisting of hydrogen, —C₁-C₁₂alkyl, —C₂-C₁₂alkenyl, —C₂-C₁₂alkynyl, —C₃-C₁₂cycloalkyl, -aryl, -heteroaryl and -heterocyclic and —R_yis selected from the group consisting of hydrogen, —C₁-C₁₂alkyl, —C₂-C₁₂alkenyl, —C₂-C₁₂alkynyl, —C₃-C₁₂cycloalkyl, -aryl, -heteroaryl, -heterocyclic, —NH₂, —NH—C₁-C₁₂alkyl, —NH—C₂-C₁₂alkenyl, —NH—C₂-C₁₂-alkynyl, —NH—C₃-C₁₂cycloalkyl, —NH-aryl, —NH-heteroaryl and —NH-heterocyclic. It is understood that the aryls, heteroaryls, alkyls, and the like can be further substituted.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The term “haloalkyl” as used herein refers to an alkyl group having 1 to (2n+1) substituent(s) independently selected from F, Cl, Br or I, where n is the maximum number of carbon atoms in the alkyl group. It will be understood that haloalkyl is a specific example of an optionally substituted alkyl.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical —OH.

As will be understood by the skilled artisan, “H” is the symbol for hydrogen, “N” is the symbol for nitrogen, “S” is the symbol for sulfur, and “O” is the symbol for oxygen. “Me” is an abbreviation for methyl.

The compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers. The term “stereoisomers” when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols “(+),” “(−),” “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present disclosure encompasses various stereoisomers of disclosed compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

The compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol

denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond.

Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both “Z” and “E” isomers. Substituents around a carbocyclic or heterocyclic ring may also be referred to as “cis” or “trans”, where the term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.”

Individual enantiomers and diastereomers of disclosed compounds can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim, 2009. Where a particular compound is described or depicted, it is intended to encompass that chemical structure as well as tautomers of that structure.

The term “enantiomerically pure” means a stereomerically pure composition of a compound. For example, a stereochemically pure composition is a composition that is free or substantially free of other stereoisomers of that compound. In another example, for a compound having one chiral center, an enantiomerically pure composition of the compound is free or substantially free of the other enantiomer. In yet another example, for a compound having two chiral centers, an enantiomerically pure composition is free or substantially free of the other diastereomers.

Where a particular stereochemistry is described or depicted it is intended to mean that a particular enantiomer is present in excess relative to the other enantiomer. A compound has an R-configuration at a specific position when it is present in excess compared to the compound having an S-configuration at that position. A compound has an S-configuration at a specific position when it is present in excess compared to the compound having an R-configuration at that position.

The compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and both solvated and unsolvated forms are contemplated herein. In one embodiment, a disclosed compound may be amorphous or a single polymorph. In another embodiment, a disclosed compound is a mixture of polymorphs. In another embodiment, a disclosed compound is in a crystalline form.

Isotopically labeled compounds are also contemplated herein, which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example, a disclosed compound may have one or more H atom replaced with deuterium.

Certain isotopically-labeled disclosed compounds (e.g., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

In some embodiments one or more of the nitrogen atoms of a disclosed compound if present are oxidized to N-oxide.

Representative synthetic routes for the preparation of the compounds disclosed herein are provided throughout the Examples section. As will be understood by the skilled artisan, diastereomers can be separated from the reaction mixture using column chromatography.

Disclosed compounds may also be prepared in embodiments, using methods described in the literature, including, but not limited to, J. Med. Chem. 2011, 54(13), 4350-64; Russian Journal of Organic Chemistry, 2011, 47(8), 1199-1203; U.S. Patent Application Publication No. 2009/0036451 A1; WO2008/046072 A2, and U.S. Pat. No. 4,336,264, the contents of each of which are expressly incorporated by reference herein.

As discussed above, contemplated herein in an embodiment is a method of increasing CFTR activity in a subject comprising administering an effective amount of a disclosed compound. Also contemplated herein is a method of treating a patient suffering from a condition associated with CFTR activity comprising administering to said patient an effective amount of a compound described herein. In certain embodiments, the disease is cystic fibrosis.

“Treating” or “treatment” includes preventing or delaying the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. A “subject” is an animal to be treated or in need of treatment. A “patient” is a human subject in need of treatment.

An “effective amount” refers to that amount of an agent that is sufficient to achieve a desired and/or recited effect. In the context of a method of treatment, an “effective amount” of the therapeutic agent that is sufficient to ameliorate of one or more symptoms of a disorder and/or prevent advancement of a disorder, cause regression of the disorder and/or to achieve a desired effect.

The term “modulating” encompasses increasing, enhancing, inhibiting, decreasing, suppressing, and the like. The terms “increasing” and “enhancing” mean to cause a net gain by either direct or indirect means. As used herein, the terms “inhibiting” and “decreasing” encompass causing a net decrease by either direct or indirect means.

In some examples, CFTR activity is enhanced after administration of a compound described herein when there is an increase in the CFTR activity as compared to that in the absence of the administration of the compound. CFTR activity encompasses, for example, chloride channel activity of the CFTR, and/or other ion transport activity (for example, HCO₃ ⁻ transport). In certain of these embodiments, the activity of one or more (e.g., one or two) mutant CFTRs (e.g., ΔF508, S549N, G542X, G551D, R117H, N1303K, W1282X, R553X, 621+1G>T, 1717-1G>A, 3849+10kbC>T, 2789+5G>A, 3120+1G>A, I507del, R1162X, 1898+1G>A, 3659delC, G85E, D1152H, R560T, R347P, 2184insA, A455E, R334W, Q493X, and 2184delA CFTR) is enhanced (e.g., increased). Contemplated patients may have a CFTR mutation(s) from one or more classes, such as without limitation, Class I CFTR mutations, Class II CFTR mutations, Class III CFTR mutations, Class IV CFTR mutations, Class V CFTR mutations, and Class VI mutations. Contemplated subject (e.g., human subject) CFTR genotypes include, without limitation, homozygote mutations (e.g., ΔF508/ΔF508 and R117H/R117H) and compound heterozygote mutations (e.g., ΔF508/G551D; ΔF508/A455E; ΔF508/G542X; Δ508F/W1204X; R553X/W1316X; W1282X/N1303K, 591Δ18/E831X, F508del/R117H/N1303K/3849+10kbC>T; Δ303K/384; and DF508/G178R).

In certain embodiments, the mutation is a Class I mutation, e.g., a G542X; a Class II/I mutation, e.g., a ΔF508/G542X compound heterozygous mutation. In other embodiments, the mutation is a Class III mutation, e.g., a G551D; a Class II/Class III mutation, e.g., a ΔF508/G551D compound heterozygous mutation. In still other embodiments, the mutation is a Class V mutation, e.g., a A455E; Class II/Class V mutation, e.g., a ΔF508/A455E compound heterozygous mutation. Of the more than 1000 known mutations of the CFTR gene, ΔF508 is the most prevalent mutation of CFTR which results in misfolding of the protein and impaired trafficking from the endoplasmic reticulum to the apical membrane (Dormer et al. (2001), J Cell Sci 114, 4073-4081; http://www.genet.sickkids.on.ca/app). In certain embodiments, ΔF508 CFTR activity is enhanced (e.g., increased). In certain embodiments, ΔF508 CFTR activity and/or G542X CFTR activity and/or G551D CFTR activity and/or A455E CFTR activity is enhanced (e.g., increased). An enhancement of CFTR activity can be measured, for example, using literature described methods, including for example, Ussing chamber assays, patch clamp assays, and hBE Ieq assay (Devor et al. (2000), Am J Physiol Cell Physiol 279(2): C461-79; Dousmanis et al. (2002), J Gen Physiol 119(6): 545-59; Bruscia et al. (2005), PNAS 103(8): 2965-2971).

As discussed above, a method of treating cystic fibrosis is also provided comprising administering a disclosed compound. Methods of treating other conditions associated with CFTR activity, including conditions associated with deficient CFTR activity are in an embodiment, contemplated herein.

In some embodiments, the disclosure is directed to a method of treating a condition associated with deficient or decreased CFTR activity comprising administering an effective amount of a disclosed compound e.g., of Formula (IIIa), (III), or (IV) that enhances CFTR activity. Non-limiting examples of conditions associated with deficient CFTR activity are cystic fibrosis, congenital bilateral absence of vas deferens (CBAVD), acute, recurrent, or chronic pancreatitis, disseminated bronchiectasis, asthma, allergic pulmonary aspergillosis, smoking-related lung diseases, such as chronic obstructive pulmonary disease (COPD), chronic sinusitis, dry eye disease, protein C deficiency, Aβ-lipoproteinemia, lysosomal storage disease, type 1 chylomicronemia, mild pulmonary disease, lipid processing deficiencies, type 1 hereditary angioedema, coagulation-fibrinolyis, hereditary hemochromatosis, CFTR-related metabolic syndrome, chronic bronchitis, constipation, pancreatic insufficiency, hereditary emphysema, and Sjogren's syndrome.

In some embodiments, disclosed methods of treatment further comprise administering an additional therapeutic agent. For example, in an embodiment, provided herein is a method of administering a disclosed compound and at least one additional therapeutic agent. In certain aspects, a disclosed method of treatment comprises administering a disclosed compound, and at least two additional therapeutic agents. Additional therapeutic agents include, for example, mucolytic agents, bronchodilators, antibiotics, anti-infective agents, anti-inflammatory agents, ion channel modulating agents, therapeutic agents used in gene therapy, CFTR correctors, and CFTR potentiators, or other agents that modulates CFTR activity. In some embodiments, at least one additional therapeutic agent is selected from the group consisting of a CFTR corrector and a CFTR potentiator. Non-limiting examples of CFTR correctors and potentiators include VX-770 (Ivacaftor), VX-809 (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid, VX-661 (1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl]-cyclopropanecarboxamide), VX-983, VX-152, VX-440, and Ataluren (PTC124) (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), FDL169, GLPG1837/ABBV-974 (for example, a CFTR potentiator), GLPG 2665, GLPG2222 (for example, a CFTR corrector); and compounds described in, e.g., WO2014/144860 and 2014/176553, hereby incorporated by reference. Non-limiting examples of modulators include QBW-251, QR-010, NB-124, and compounds described in, e.g., WO2014/045283; WO2014/081821, WO2014/081820, WO2014/152213; WO2014/160440, WO2014/160478, US2014027933; WO2014/0228376, WO2013/038390, WO2011/113894,WO2013/038386; and WO2014/180562, of which the disclosed modulators in those publications are contemplated as an additional therapeutic agent and incorporated by reference. Non-limiting examples of anti-inflammatory agents include N6022 (3-(5-(4-(1H-imidazol-1-yl) phenyl)-1-(4-carbamoyl-2-methylphenyl)-¹H-pyrrol-2-yl) propanoic acid), CTX-4430, N1861, N1785, and N91115.

In some embodiments, the methods described herein can further include administering an additional therapeutic agent or administering at least two additional CFTR therapeutic agents. In some embodiments, the methods described herein can further include administering an additional CFTR modulator or administering at least two additional CFTR modulators. In certain embodiments, at least one CFTR modulator is a CFTR corrector (e.g., VX-809, VX-661, VX-983, VX-152, VX-440, GLPG2222 and GLPG2665) or potentiator (e.g., ivacaftor, genistein and GLPG 1837). In certain of these embodiments, one of the at least two additional therapeutic agents is a CFTR corrector (e.g., VX-809, VX-661,VX-983, VX-152, and VX-440) and the other is a CFTR potentiator (e.g., ivacaftor and genistein). In certain of these embodiments, one of the at least two additional therapeutic agents is a CFTR corrector (e.g., GLPG2222 or GLPG2665) and the other is a CFTR potentiator (e.g., GLPG1837). In certain of these embodiments, one of the at least two additional therapeutic agents is a CFTR corrector (e.g., VX-809 or VX-661) and the other is a CFTR potentiator (e.g., ivacaftor). In certain of these embodiments, at least one CFTR modulator is an agent that enhances readthrough of stop codons (e.g., NB124 or ataluren).

Accordingly, in another aspect, this disclosure provides a method of treating a condition associated with deficient or decreased CFTR activity (e.g., cystic fibrosis), which includes administering to a subject in need thereof (e.g., a human patient in need thereof) an effective amount of a disclosed compound and at least one or two additional CFTR therapeutic agent(s) (e.g., at least one or two additional CFTR therapeutic agents, e.g., in which one of the at least one or two additional therapeutic agents is optionally a CFTR corrector or modulator (e.g., VX-809, VX-661, VX-983, VX-152, VX-440, GLPG2222, GLPG2665, NB124, ataluren) and/or the other is a CFTR potentiator (e.g., ivacaftor, genistein, and GLPG1837); e.g., one of the at least two additional therapeutic agents is GLPG2222 or GLPG2665, and the other is GLPG1837; or one of the at least two additional therapeutic agents is VX-809 or VX-661, and the other is a ivacaftor). In certain embodiments, the subject's CFTR genotype includes, without limitation, one or more Class I CFTR mutations, one or more Class II CFTR mutations, one or more Class III CFTR mutations, one or more Class IV CFTR mutations, or one or more Class V CFTR mutations, or one or more Class VI CFTR mutations. In certain embodiments, the subject's CFTR genotype includes, without limitation, one or more homozygote mutations (e.g., ΔF508/ΔF508 or R117H/R117H) and/or one or more compound heterozygote mutations (e.g., ΔF508/G551D; ΔF508/A455E; ΔF508/G542X; Δ508F/W1204X; R553X/W1316X; W1282X/N1303K; F508del/R117H; N1303K/3849+10kbC>T; ΔF508/R334W; DF508/G178R, and 591Δ18/E831X). In certain embodiments, the subject's CFTR genotype includes a Class I mutation, e.g., a G542X Class I mutation, e.g., a ΔF508/G542X compound heterozygous mutation. In other embodiments, the subject's CFTR genotype includes a Class II mutation, e.g., a G551D Class III mutation, e.g., a ΔF508/G551D compound heterozygous mutation. In still other embodiments, the subject's CFTR genotype includes a Class V mutation, e.g., a A455E Class V mutation, e.g., a ΔF508/A455E compound heterozygous mutation. In certain embodiments, ΔF508 CFTR activity and/or G542X CFTR activity and/or G551D CFTR activity and/or A455E activity is enhanced (e.g., increased). In certain embodiments, the enhancement in activity (e.g., increase in activity) provided by the combination of the disclosed compound and one or two additional therapeutic agents is greater than additive when compared to the enhancement in activity provided by each therapeutic component individually.


Class	Effect on CFTR protein	Example of mutation

I	Shortened protein	W1282X Instead of inserting the
		amino acid tryptophan (W), the
		protein sequence is prematurely
		stopped (indicated by an X).
II	Protein fails to	ΔF508 A phenylalanine amino
	reach cell membrane	acid (F) is deleted
III	Channel cannot be	G551D A “missense” mutation:
	regulated properly	instead of a glycine amino acid
		(G), aspartate (D) is added
IV	Reduced chloride conductance	R117H Missense
V	Reduced due to incorrect	3120 + 1G > A Splice-site
	splicing of gene	mutation in gene intron 16
VI	Reduced due to protein	N287Y a A −>T at 991
	instability


Genotype	Description	Possible Symptoms

Δ508F/Δ508F	homozygote	Severe lung disease,
		pancreatic insufficient
R117H/R117H	homozygote	Congenital bilateral absence
		of the vas deferens,
		No lung or pancreas disease,
WT/Δ508F	heterozygote	Unaffected
WT/3120 + 1 G > A	heterozygote	Unaffected
Δ508F/W1204X	compound	No lung disease, pancreatic
	heterozygote	insufficient
R553X and W1316X	compound	Mild lung disease,
	heterozygote	pancreatic insufficient
591Δ18/E831X	compound	No lung or pancreas disease,
	heterozygote	nasal polyps

For example, provided herein is a method of treating a patient having one or more of the following mutations in the CFTR gene: G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, S549R, G970R, or R117H, and/or e.g., a patient with one or two copies of the F508del mutation, or one copy of the ΔF508 mutation and a second mutation that results in a gating effect in the CFTR protein (e.g., a patient that is heterozygous for ΔF508 and G551D mutation), a patient with one copy of the ΔF508 mutation and a second mutation that results in residual CFTR activity, or a patient with one copy of the ΔF508 mutation and a second mutation that results in residual CFTR activity, comprising administering an effective amount of a disclosed compound. As described herein, such exemplary methods (e.g., of a patient having one or mutations such as those described above) may include, for example, administering to such patient a combination therapy, e.g., administering (simultaneously or sequentially) an effective amount of ivacaftor to said patient and an effective amount of disclosed compound that may act as an amplifier. Such administration may result, for example, in increased chloride transport in human bronchial epithelial cells with e.g., one or two copies of mutations, e.g, ΔF508 mutation, as compared to administration of ivacaftor alone. Another combination therapy that includes a disclosed compound may also include an effective amount of a readthrough agent (e.g., ataluren, NB124) and an effect amount of disclosed compound that may act as an amplifier.

The phrase “combination therapy,” as used herein, refers to an embodiment where a patient is co-administered a disclosed compound, a CFTR potentiator agent (e.g., ivacaftor) and optionally, one or more CFTR corrector agent(s) (e.g, VX-661 and/or lumacaftor) as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents. For example, a beneficial effect of a combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. For example, administration of a disclosed compound with ivacaftor alone or with a CFTR corrector agent (e.g., lumacaftor or VX-661) may result in a level of function (e.g., as measured by chloride activity in HBE cells or patients that have a ΔF508 mutation) that achieves clinical improvement (or better) as compared to the chloride activity level in cells or patients with a G551D mutation receiving ivacaftor alone, or ivacaftor and a corrector agent (lumacaftor or VX-661); or for example, administration of a disclosed compound with ivacaftor alone or ivacaftor with a CFTR corrector agent (e.g., lumacaftor or VX-661) may result in a level of function (e.g., as measured by chloride activity in HBE cells or patients that have a A455E mutation, that achieves clinical improvement (or better) as compared to the chloride activity level at e.g., 50% or more of wild type cells; or upon administration of a disclosed compound and ivacaftor to a patient (e.g. having a G551D class III mutation) may show e.g., about two times or more improved activity of ivacaftor as compared to administration of ivacaftor alone. Administration of disclosed therapeutic agents in combination typically is carried out over a defined time period (usually a day, days, weeks, months or years depending upon the combination selected). Combination therapy is intended to embrace administration of multiple therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single tablet or capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, inhalational routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection or inhalation or nebulizer while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection, inhalation or nebulization.

Combination therapy also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies. Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by a day, days or even weeks.

The components of a disclosed combination may be administered to a patient simultaneously or sequentially. It will be appreciated that the components may be present in the same pharmaceutically acceptable carrier and, therefore, are administered simultaneously. Alternatively, the active ingredients may be present in separate pharmaceutical carriers, such as, conventional oral dosage forms, that can be administered either simultaneously or sequentially.

The term “pharmaceutically acceptable salt(s)” as used herein refers to salts of acidic or basic groups that may be present in a disclosed compounds used in disclosed compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids. The compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.

In an embodiment, methods provided herein may include administering prodrugs of the compounds described herein, for example, prodrugs of a compound of Formula (IIIa), (III), or (IV), or a pharmaceutical composition thereof or method of use of the prodrug.

The term “prodrug” refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). For example, if a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C_1-8)alkyl, (C_2-12)alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N—(C_1-2)alkylamino(C_2-3)alkyl (such as β-dimethylaminoethyl), carbamoyl-(C_1-2)alkyl, N,N-di(C_1-2)alkylcarbamoyl-(C_1-2)alkyl and piperidino-, pyrrolidino- or morpholino(C_2-3)alkyl.

Similarly, if a disclosed compound contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C_1-6)alkylcarbonyloxymethyl, 1-((C_1-6)alkylcarbonyloxy)ethyl, 1-methyl-1-((C_1-6)alkylcarbonyloxy)ethyl (C_1-6)alkoxycarbonyloxymethyl, N—(C_1-6)alkoxycarbonylaminomethyl, succinoyl, (C_1-6)alkylcarbonyl, α-amino(C_1-4)alkylcarbonyl, arylalkylcarbonyl and α-aminoalkylcarbonyl, or α-aminoalkylcarbonyl-α-aminoalkylcarbonyl, where each α-aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)₂, —P(O)(O(C_1-6)alkyl)₂or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

If a disclosed compound incorporates an amine functional group, a prodrug can be formed, for example, by creation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine. In addition, a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine. For examples, see Simplicio, et al., Molecules 2008, 13, 519 and references therein

Also contemplated in certain embodiments is the use of clathrates of the compounds described herein, pharmaceutical compositions comprising the clathrates, and methods of use of the clathrates. Clathrates of a disclosed compound, e.g, Formula (IIIa), (III), or (IV), or a pharmaceutical composition thereof are contemplated.

As discussed above, the disclosure also contemplates administration of pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and a compound described herein. A disclosed compound, or a pharmaceutically acceptable salt, solvate, clathrate or prodrug thereof, can be administered in pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient. The excipient can be chosen based on the expected route of administration of the composition in therapeutic applications. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder and oral administration may be preferred to treat a gastrointestinal disorder. The route of administration and the dosage of the composition to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. A pharmaceutical composition comprising a disclosed compound or a pharmaceutically acceptable salt, solvate, clathrate or prodrug, can be administered by a variety of routes including, but not limited to, parenteral, oral, pulmonary, ophthalmic, nasal, rectal, vaginal, aural, topical, buccal, transdermal, intravenous, intramuscular, subcutaneous, intradermal, intraocular, intracerebral, intralymphatic, intraarticular, intrathecal and intraperitoneal. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the pharmacologic agent or composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SEPHAROSE™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

Disclosed compositions may be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating a composition into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Parenteral formulations may also include antibacterial agents such as, for example, benzyl alcohol or methyl parabens, antioxidants such as, for example, ascorbic acid or sodium bisulfite and chelating agents such as EDTA. Buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

Injectable formulations can be prepared either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can also be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above [Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997]. The compositions and pharmacologic agents described herein can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, transdermal applications and ocular delivery. For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1% to about 2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Topical application can result in transdermal or intradermal delivery. Transdermal delivery can be achieved using a skin patch or using transferosomes. [Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998].

For the purpose of oral therapeutic administration, the pharmaceutical compositions can be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. Tablets, pills, capsules, troches and the like may also contain binders, excipients, disintegrating agent, lubricants, glidants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used. In another embodiment, the composition is administered as a tablet or a capsule.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor, and the like. For vaginal administration, a pharmaceutical composition may be presented as pessaries, tampons, creams, gels, pastes, foams or spray.

The pharmaceutical composition can also be administered by nasal administration. As used herein, nasally administering or nasal administration includes administering the composition to the mucus membranes of the nasal passage or nasal cavity of the patient. As used herein, pharmaceutical compositions for nasal administration of a composition include therapeutically effective amounts of the compounds prepared by well-known methods to be administered, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream or powder. Administration of the composition may also take place using a nasal tampon or nasal sponge.

For topical administration, suitable formulations may include biocompatible oil, wax, gel, powder, polymer, or other liquid or solid carriers. Such formulations may be administered by applying directly to affected tissues, for example, a liquid formulation to treat infection of conjunctival tissue can be administered dropwise to the subject's eye, or a cream formulation can be administered to the skin.

Rectal administration includes administering the pharmaceutical compositions into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can easily be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120° C., dissolving the pharmaceutical composition in the glycerin, mixing the heated glycerin after which purified water may be added, and pouring the hot mixture into a suppository mold.

Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves and the like.

In addition to the usual meaning of administering the formulations described herein to any part, tissue or organ whose primary function is gas exchange with the external environment, for purposes of the present invention, “pulmonary” will also mean to include a tissue or cavity that is contingent to the respiratory tract, in particular, the sinuses. For pulmonary administration, an aerosol formulation containing the active agent, a manual pump spray, nebulizer or pressurized metered-dose inhaler as well as dry powder formulations are contemplated. Suitable formulations of this type can also include other agents, such as antistatic agents, to maintain the disclosed compounds as effective aerosols.

A drug delivery device for delivering aerosols comprises a suitable aerosol canister with a metering valve containing a pharmaceutical aerosol formulation as described and an actuator housing adapted to hold the canister and allow for drug delivery. The canister in the drug delivery device has a head space representing greater than about 15% of the total volume of the canister. Often, the compound intended for pulmonary administration is dissolved, suspended or emulsified in a mixture of a solvent, surfactant and propellant. The mixture is maintained under pressure in a canister that has been sealed with a metering valve.

The disclosure also encompasses the treatment of a condition associated with a dysfunction in proteostasis in a subject comprising administering to said subject an effective amount of a disclosed compound that enhances, improves or restores proteostasis of a protein. Proteostasis refers to protein homeostasis. Dysfunction in protein homeostasis is a result of protein misfolding, protein aggregation, and defective protein trafficking or protein degradation. For example, the disclosure in some embodiments contemplates administering a disclosed compound that corrects protein misfolding, reduces protein aggregation, corrects or restores protein trafficking and/or affects protein degradation for the treatment of a condition associated with a dysfunction in proteostasis. For example, a disclosed compound may corrects protein misfolding and/or corrects or restores protein trafficking is administered. In cystic fibrosis, the mutated or defective enzyme is the cystic fibrosis transmembrane conductance regulator (CFTR). One of the most common mutations of this protein is ΔF508 which is a deletion (Δ) of three nucleotides resulting in a loss of the amino acid phenylalanine (F) at the 508th (508) position on the protein. As described above, mutated cystic fibrosis transmembrane conductance regulator exists in a misfolded state and is characterized by altered trafficking as compared to the wild type CFTR. Additional exemplary proteins of which there can be a dysfunction in proteostasis, for example that can exist in a misfolded state, include, but are not limited to, glucocerebrosidase, hexosamine A, aspartylglucsaminidase, α-galactosidase A, cysteine transporter, acid ceremidase, acid α-L-fucosidase, protective protein, cathepsin A, acid β-glucosidase, acid β-galactosidase, iduronate 2-sulfatase, α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acid β-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid β-galactosidase, N-acetylglucosamine-1-phosphotransferase, acid sphingmyelinase, NPC-1, acid α-glucosidase, β-hexosamine B, heparin N-sulfatase, α-N-acetylglucosaminidase, α-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, α-N-acetylgalactosaminidase, α-neuramidase, β-glucuronidase, β-hexosamine A and acid lipase, polyglutamine, α-synuclein, TDP-43, superoxide dismutase (SOD), Aβ peptide, tau protein, transthyretin and insulin. The compounds of e.g., Formula (IIIa), (III), or (IV) may be used to restore proteostasis (e.g., correct folding and/or alter trafficking) of the proteins described above.

Protein conformational diseases encompass gain of function disorders and loss of function disorders. In one embodiment, the protein conformational disease is a gain of function disorder. The terms “gain of function disorder,” “gain of function disease,” “gain of toxic function disorder” and “gain of toxic function disease” are used interchangeably herein. A gain of function disorder is a disease characterized by increased aggregation-associated proteotoxicity. In these diseases, aggregation exceeds clearance inside and/or outside of the cell. Gain of function diseases include, but are not limited to, neurodegenerative diseases associated with aggregation of polyglutamine, Lewy body diseases, amyotrophic lateral sclerosis, transthyretin-associated aggregation diseases, Alzheimer's disease, Machado-Joseph disease, cerebral B-amyloid angiopathy, retinal ganglion cell degeneration, tauopathies (progressive supranuclear palsy, corticobasal degeration, frontotemporal lobar degeneration), cerebral hemorrhage with amyloidosis, Alexander disease, Serpinopathies, familial amyloidotic neuropathy, senile systemic amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, familial amyloidosis of the Finnish type, lysoyzme amyloidosis, fibrinogen amyloidosis, dialysis amyloidosis, inclusion body myositis/myopathy, cataracts, medullary thyroid carcinoma, cardiac atrial amyloidosis, pituitary prolactinoma, hereditary lattice corneal dystrophy, cutaneous lichen amyloidosis, corneal lactoferrin amyloidosis, corneal lactoferrin amyloidosis, pulmonary alveolar proteinosis, odontogenic tumor amyloid, seminal vesical amyloid, sickle cell disease, critical illness myopathy, von Hippel-Lindau disease, spinocerebellar ataxia 1, Angelman syndrome, giant axon neuropathy, inclusion body myopathy with Paget disease of bone, frontotemporal dementia (IBMPFD) and prion diseases. Neurodegenerative diseases associated with aggregation of polyglutamine include, but are not limited to, Huntington's disease, dentatorubral and pallidoluysian atrophy, several forms of spino-cerebellar ataxia, and spinal and bulbar muscular atrophy. Alzheimer's disease is characterized by the formation of two types of aggregates: extracellular aggregates of Aβ peptide and intracellular aggregates of the microtubule associated protein tau. Transthyretin-associated aggregation diseases include, for example, senile systemic amyloidoses and familial amyloidotic neuropathy. Lewy body diseases are characterized by an aggregation of α-synuclein protein and include, for example, Parkinson's disease, lewy body dementia (LBD) and multiple system atrophy (SMA). Prion diseases (also known as transmissible spongiform encephalopathies or TSEs) are characterized by aggregation of prion proteins. Exemplary human prion diseases are Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease, Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia and Kuru. In another embodiment, the misfolded protein is alpha-1 anti-trypsin.

In a further embodiment, the protein conformation disease is a loss of function disorder. The terms “loss of function disease” and “loss of function disorder” are used interchangeably herein. Loss of function diseases are a group of diseases characterized by inefficient folding of a protein resulting in excessive degradation of the protein. Loss of function diseases include, for example, lysosomal storage diseases. Lysosomal storage diseases are a group of diseases characterized by a specific lysosomal enzyme deficiency which may occur in a variety of tissues, resulting in the build-up of molecules normally degraded by the deficient enzyme. The lysosomal enzyme deficiency can be in a lysosomal hydrolase or a protein involved in the lysosomal trafficking. Lysosomal storage diseases include, but are not limited to, aspartylglucosaminuria, Fabry's disease, Batten disease, Cystinosis, Farber, Fucosidosis, Galactasidosialidosis, Gaucher's disease (including Types 1, 2 and 3), Gm1 gangliosidosis, Hunter's disease, Hurler-Scheie's disease, Krabbe's disease, α-Mannosidosis, β-Mannosidosis, Maroteaux-Lamy's disease, Metachromatic Leukodystrophy, Morquio A syndrome, Morquio B syndrome, Mucolipidosis II, Mucolipidosis III, Neimann-Pick Disease (including Types A, B and C), Pompe's disease, Sandhoff disease, Sanfilippo syndrome (including Types A, B, C and D), Schindler disease, Schindler-Kanzaki disease, Sialidosis, Sly syndrome, Tay-Sach's disease and Wolman disease.

In another embodiment, the disease associated with a dysfunction in proteostasis is a cardiovascular disease. Cardiovascular diseases include, but are not limited to, coronary artery disease, myocardial infarction, stroke, restenosis and arteriosclerosis. Conditions associated with a dysfunction of proteostasis also include ischemic conditions, such as, ischemia/reperfusion injury, myocardial ischemia, stable angina, unstable angina, stroke, ischemic heart disease and cerebral ischemia.

In yet another embodiment, the disease associated with a dysfunction in proteostasis is diabetes and/or complications of diabetes, including, but not limited to, diabetic retinopathy, cardiomyopathy, neuropathy, nephropathy, and impaired wound healing.

In a further embodiment, the disease associated with a dysfunction in proteostasis is an ocular disease including, but not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, glaucoma, cataracts, retinitis pigmentosa (RP) and dry macular degeneration.

In yet additional embodiments, a disclosed method is directed to treating a disease associated with a dysfunction in proteostasis, wherein the disease affects the respiratory system or the pancreas. In certain additional embodiments, a contemplated method encompass treating a condition selected from the group consisting of polyendocrinopathy/hyperinsulinemia, diabetes mellitus, Charcot-Marie Tooth syndrome, Pelizaeus-Merzbacher disease, and Gorham's Syndrome.

Additional conditions associated with a dysfunction of proteostasis include hemoglobinopathies, inflammatory diseases, intermediate filament diseases, drug-induced lung damage and hearing loss. For example, provided herein are methods for the treatment of hemoglobinopathies (such as sickle cell anemia), an inflammatory disease (such as inflammatory bowel disease, colitis, ankylosing spondylitis), intermediate filament diseases (such as non-alcoholic and alcoholic fatty liver disease) and drug induced lung damage (such as methotrexate-induced lung damage). In another embodiment, methods for treating hearing loss, such as noise-induced hearing loss, aminoglycoside-induced hearing loss, and cisplatin-induced hearing loss comprising administering a disclosed compound are provided.

Additional conditions include those associated with a defect in protein trafficking and that can be treated include: PGP mutations, hERG trafficking mutations, nephrongenic diabetes insipidus mutations in the arginine-vasopressin receptor 2, persistent hyperinsulinemic hypoglycemia of infancy (PHH1) mutations in the sulfonylurea receptor 1, and α1AT.

The invention is illustrated by the following examples which are not meant to be limiting in any way.

EXEMPLIFICATION

The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials. At least some of the compounds identified as “intermediates” herein are contemplated as compounds of the invention.

Example 1 5-phenyl-N-((tetrahydrofuran-2-yl)methyl)thiazole-2-carboxamide

HOBt (0.11 g, 0.8 mmol) and EDC.HCl (0.20 g, 1.1 mmol) were added to a solution 5-phenylthiazole-2-carboxylic acid (0.15 g, 0.73 mmol) in THF (5 mL) followed by the addition of (tetrahydro-furan-2-yl)-methylamine (0.15 g, 1.4 mmol) and stirred at room temperature for 12 h. Volatiles were removed under vacuum and crude was dissolved in DCM. The organic layer was washed with water, brine, dried over anhydrous sodium sulfate and concentrated under vacuum to afford crude product. The crude was purified by combiflash chromatography using EtOAc in hexane to obtain the product (90 mg, 43%) as off white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.97 (s, 1H), 7.60-7.59 (m, 2H), 7.53 (brs, 1H), 7.44-7.38 (m, 3H), 4.09-4.06 (m, 1H), 3.94-3.88 (m, 1H), 3.80-3.75 (m, 1H), 3.74-3.68 (m, 1H), 3.45-3.38 (m, 1H), 2.03-1.98 (m, 1H), 1.95-1.89 (m, 2H), 1.64-1.58 (m, 1H); LC-MS: [M+H]⁺ 289.1; HPLC Purity: 99.85% at 254 nm and 99.85% at 220 nm.

Example 2 N-(2-methoxyethyl)-5-phenylthiazole-2-carboxamide

The product was prepared using the procedure described in example 1. The crude was purified by combiflash chromatography using EtOAc in hexane to obtain the product (30 mg, 16%) as white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.98 (s, 1H), 7.60-7.58 (m, 2H), 7.53 (br, 1H), 7.49-7.38 (m, 3H), 3.67-3.62 (m, 2H), 3.59-3.55 (m, 2H), 3.39 (s, 3H); LC-MS: [M+H]⁺ 263.0; HPLC Purity: 99.62% at 254 nm and 99.68% at 220 nm.

Example 3: N-(2-morpholinoethyl)-5-phenylthiazole-2-carboxamide

The product was prepared using the procedure described in example 1. The crude was purified by preparative HPLC obtain the product (85 mg, 37%) as off white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.99 (s, 1H), 7.62 (bs, 1H), 7.60-7.58 (m, 2H), 7.45-7.35 (m, 3H), 3.75-3.72 (m, 4H), 3.59-3.54 (m, 2H), 2.61-2.58 (m, 2H), 2.58-2.48 (m, 4H); LC-MS: [M+H]⁺ 317.8; HPLC Purity: 97.36% at 254 nm and 97.87% at 220 nm.

Example 4 N-(3-(1H-imidazol-1-yl)propyl)-5-phenylthiazole-2-carboxamide

The product was prepared using the procedure described in example 1. The crude was purified by preparative HPLC obtain the product (92 mg, 40%) as white solid. ¹H NMR (400 MHz, CDCl₃): δ 7.98 (s, 1H), 7.60-7.58 (d, J=6.8 Hz, 2H), 7.52 (s, 1H), 7.45-7.36 (m, 3H), 7.34-7.30 (m, 1H), 7.07 (s, 1H), 6.97 (s, 1H), 4.07-4.04 (t, J=7.0 Hz, 2H), 3.51-3.46 (m, 2H), 2.17-2.10 (m, 2H); LC-MS: [M+H]⁺312.9; HPLC Purity: 99.89% at 254 nm and 99.74% at 220 nm.

Example 5 N-(3-(1-methyl-1H-pyrazol-5-yl)propyl)-5-phenylthiazole-2-carboxamide

The product was prepared using the procedure described in example 1. The crude was purified by column chromatography using flash silica gel eluted with 50% EtOAc in hexane to afford (0.115 g, 36.27%) as off white solid; ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.60-7.58 (m, 2H), 7.45-7.37 (m, 4H), 7.30 (br, 1H), 6.06 (s, 1H), 3.79 (s, 3H), 3.58-3.53 (q, 2H), 2.72-2.68 (t, 2H), 2.04-1.96 (m, 2H); LCMS [M+H]⁺ 327.1; HPLC purity: 92.12% at 220 nm and 98.02% at 254 nm.

Example 6 N-(2-(5-(hydroxymethyl)-1,3,4-thiadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide

Step 1: ethyl 2-(2-(3-((tert-butoxycarbonyl)amino)propanoyl)hydrazinyl)-2-oxoacetate: Et₃N (4.98 g, 49.261 mmol) was added to an ice-cooled solution of tert-butyl (3-hydrazinyl-3-oxopropyl)carbamate (5.0 g, 24.63 mmol) in DCM (100 mL) followed by ethyl oxalyl chloride (4.01 g, 29.55 mmol) and the resulting reaction mixture was stirred at room temperature for 2 h. Then the reaction mixture was diluted with water (50 mL). Organic layer separated off and the aq. layer was further extracted with DCM (50 mL×2). Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the product (7 g, crude) as yellowish sticky mass which was used as such in next step without further purification. As per 1H-NMR, triethyl amine present as an impurity in the compound. LC-MS: [M+H]⁺=303.9.

Step 2: ethyl 5-(2-((tert-butoxycarbonyl)amino)ethyl)-1,3,4-thiadiazole-2-carboxylate: Lawesson's reagent (9.34 g, 23.10 mmol) was added to a solution of ethyl 2-(2-(3-((tert-butoxycarbonyl)amino)propanoyl)hydrazinyl)-2-oxoacetate (7 g, crude) in 1,4-dioxane (150 mL) and the reaction mixture was refluxed for 2h. Then the reaction mixture was cooled, neutralized with saturated sodium carbonate solution and extracted with EtOAc (100 mL×3). Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude product. The crude compound thus obtained was purified by silica gel of (100-200 mesh) column chromatography using 20% EtOAc in Hexane as eluent to obtain the product (2.5 g, 33.61% after two steps) as yellow viscous mass. ¹H NMR (400 MHz, CDCl₃): δ 5.02 (br, 1H), 4.51 (q, J=7.2 Hz, 2H), 3.61-3.60 (m, 2H), 3.39-3.37 (t, J=6.3 Hz, 2H), 1.44 (t, 3H) 1.41 (s, 9H); LC-MS: [M+H]⁺=302.0.

Step 3: ethyl 5-(2-aminoethyl)-1,3,4-thiadiazole-2-carboxylate hydrochloride: 4M HCl in 1,4-dioxane (3 mL) was added to an ice-cooled solution of ethyl 5-(2-((tert-butoxycarbonyl)amino)ethyl)-1,3,4-thiadiazole-2-carboxylate (0.200 g, 0.66 mmol) in dioxane (5 mL) and the resulting reaction mixture was stirred at room temperature for 2 h. Volatiles were removed under reduced pressure to obtain the product (0.150 g, crude) as yellowish solid which was used as such for coupling. LC-MS: [M+H]⁺=202.2.

Step 4: ethyl 5-(2-(5-phenylthiazole-2-carboxamido)ethyl)-1,3,4-thiadiazole-2-carboxylate: HATU (0.278 g, 0.73 mmol) and DIPEA (0.188 g, 1.46 mmol) were added sequentially to a solution of 5-phenylthiazole-2-carboxylic acid (0.100 g, 0.48 mmol) in THF (10 mL) and the reaction mixture was stirred for 10 min. Ethyl 5-(2-aminoethyl)-1,3,4-thiadiazole-2-carboxylate hydrochloride (0.147 g, crude) was then added and the reaction mixture was stirred at room temperature for 12 h. Volatiles were removed under vacuum and the crude compound was dissolved in DCM. The organic layer was washed with water, brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude product. The crude compound was purified by silica gel (100-200 mesh) column chromatography using 20% EtOAc in Hexane as eluent to obtain the product (0.150 g, 63%) as off white solid. ¹H NMR (400 MHz, DMSO): δ 9.13 (t, J=5.9 Hz, 1H), 8.42 (s, 1H), 7.77 (d, J=8.5 Hz 2H), 7.50-7.49 (m, 2H), 7.48-7.44 (m, 1H), 4.40 (q, J=7.1 Hz, 2H), 3.71 (q, J=6.3 Hz, 2H), 3.49 (t, J=6.5 Hz, 2H), 1.32 (t, J=7.12 Hz, 3H); LC-MS: [M+H]⁺=388.8.

Step 5: N-(2-(5-(hydroxymethyl)-1,3,4-thiadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide: Sodium borohydride (0.063 g, 1.67 mmol) was added in portions to an ice-cooled solution of ethyl 5-(2-(5-phenylthiazole-2-carboxamido)ethyl)-1,3,4-thiadiazole-2-carboxylate (0.130 g, 0.33 mmol) in ethanol (20 mL) and the reaction mixture was stirred for 2 h at room temperature. Volatiles were removed under reduced pressure and the reaction mixture was diluted with cold water. The aq. layer was extracted with EtOAc (30 mL×3). Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford crude product. The crude compound thus obtained was purified by silica gel (100-200 mesh) column chromatography using 70% EtOAc in Hexane as eluent to obtain the product (0.050 g, 43.47%) as white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.09 (t, J=5.8 Hz, 1H), 8.42 (s, 1H), 7.77 (dd, 2H), 7.50-7.46 (m, 2H), 7.43-7.41 (m, 1H), 6.13 (t, J=5.9 Hz, 1H), 4.79 (d, J=5.9 Hz, 2H), 3.67 (q, J=6.5 Hz, 2H), 3.39-3.35 (m, 2H); LC-MS: [M+H]⁺=347.0; HPLC purity: 99.27% at 220 nm and 99.85% at 254 nm.

Example 7 N-(2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide

Step 1: 3-(1,3-dioxoisoindolin-2-yl)-N′-(2-methoxyacetyl)propanehydrazide: EDC.HCl (2.8 g, 15.0 mmol) was added to a solution of 3-(1,3-dioxoisoindolin-2-yl)propanoic acid (2.2 g, 10.0 mmol) in THF (30 mL followed by HOBt (1.84 g, 12.0 mmol) and the reaction mixture was stirred for 20 min at room temperature. 2-methoxyacetohydrazide (1.05 g, 10.0 mmol) was added to the reaction mixture followed by DIPEA (6.4 g, 50.0 mmol) and the reaction mixture was stirred at room temperature for 16 h. Volatiles were removed under reduced pressure and the reaction mixture was diluted with water (30 mL). The aq. layer was extracted with DCM (3×25 mL), combined organic layer was washed with aqueous NaHCO₃solution (50 mL), dried over anhydrous Na₂SO₄and concentrated under reduced pressure to obtain the product (2.0 g, 65%) as white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.91 (s, 1H), 9.72 (s, 1H), 7.88-7.82 (m, 4H), 3.88 (s, 2H), 3.78 (t, J=7.6 Hz, 2H), 3.30 (s, 3H), 2.25 (t, J=7.6 Hz, 2H); LC-MS: [M+H]⁺=306.3 m/z.

Step 2: 2-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)isoindoline-1,3-dione: T₃P (10 mL, 15.7 mmol) was added to a solution of 3-(1,3-dioxoisoindolin-2-yl)-N′-(2-methoxyacetyl)propanehydrazide (2.0 g, 6.55 mmol) in 1, 4-dioxane (60 mL) and the reaction mixture was heated to 100° C. for 18 h. Volatiles were removed under reduced pressure and the crude reaction mixture was diluted with aqueous NaHCO₃solution (50 mL). The aq. layer was extracted with EtOAc (2×50 mL). Combined organic layer was washed with water (50 mL), brine (50 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the product (1.4 g, 74%) as off-white solid which was used as such without further purification. ¹H NMR (400 MHz, DMSO-d₆): δ 7.89-7.83 (m, 4H), 4.57 (s, 2H), 3.94 (t, J=7.6 Hz, 2H), 3.28 (s, 3H), 3.22 (t, J=7.6 Hz, 2H); LC-MS: [M+H]⁺=288.2 m/z.

Step 3: 2-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)isoindoline-1,3-dione: hydrazine hydrate (0.4 mL) was added to a solution of 2-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)isoindoline-1,3-dione (0.75 g, 2.6 mmol) in ethanol (10 mL) and the reaction mixture was stirred at room temperature for 16 h. Then the reaction mixture was filtered and concentrated under reduced pressure to obtain the product (0.32 g, crude) which was used as such in next step without further purification. LC-MS: [M+H]⁺=157.9 m/z.

Step 4: N-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide: A solution of 2-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)isoindoline-1,3-dione (0.314 g, crude) and ethyl 5-phenylthiazole-2-carboxylate (0.29 g, 1.2 mmol) in DMF (5 mL) was heated to 65° C. for 16 h. After completion, the reaction mixture was diluted with water (25 mL) and extracted with DCM (2×25 mL). Combined organic layer was washed with water (30 mL) followed by brine (30 mL) and dried over sodium sulfate. Solvent was removed under reduced pressure to afford crude product. The crude compound was purified by combiflash to obtain the product (0.14 g) as off-white solid. LC-MS: [M+H]⁺=345.1 m/z. As per 1H-NMR, compound is impure and used as such in next step without further purification.

Step 5: N-(2-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide: BBr₃(0.15 ml, 1.56 mmol) was added to a −78° C. solution of N-(2-(5-(methoxymethyl)-1,3,4-oxadiazol-2-yl)ethyl)-5-phenylthiazole-2-carboxamide (0.140 g, crude) in DCM (6 mL) and the reaction mixture was stirred at room temperature for 5 h. Then the reaction mixture was poured onto ice, stirred for 10 min. The aq. layer was basified with saturated sodium bicarbonate solution and extracted with DCM (2×20 mL). Combined organic layer was washed with water, dried over sodium sulfate and concentrated under reduced pressure to afford crude product. The crude compound thus obtained was purified by crystallization to obtain the product (35 mg, 4.3% over three steps) as white crystal. ¹H NMR (400 MHz, DMSO-d₆): δ 9.07 (t, J=6.0 Hz, 1H), 8.42 (s, 1H), 7.77 (d, J=7.2 Hz, 2H), 7.48 (m, 2H), 7.42 (m, 2H), 5.86 (t, J=6.0 Hz, 1H), 4.59 (d, J=6.0 Hz, 2H), 3.66 (q, J=6.7 Hz, 2H), 3.14 (t, J=7.2 Hz, 2H); LC-MS: [M+H]⁺=331.1 m/z; HPLC purity 98.02% at 314 nm, 97.60% at 254 nm.

Examples 8 and 9 N-(2-(4-(hydroxymethyl)-5-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide and N-(2-(5-(hydroxymethyl)-4-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide

Step 1: N-(2-(4-(hydroxymethyl)-5-methyl-H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide and N-(2-(5-(hydroxymethyl)-4-methyl-1H-1,2,3-trazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide: a solution of 2-(2-azidoethyl)isoindoline-1,3-dione (5.0 g, 23.0 mmol) and 2-butyn-1-ol (2.0 mL, 26.7 mmol) in toluene (50 mL) was heated to 110° C. in a sealed tube and for 48 h. After completion, volatiles were removed under reduced pressure and crude reaction mixture thus obtained was washed with ether (100 mL). Residue was dried to obtain (4.5 g, 68%) as a mixture of two regioisomers which was used as such in the next step without further purification. ¹H NMR (400 MHz, DMSO-d₆): δ 7.86-7.82 (m, 8H), 5.36 (t, J=5.2 Hz, 1H), 5.00 (t, J=5.2 Hz, 1H), 4.59 (t, J=5.6 Hz, 2H), 4.56 (t, J=5.2 Hz, 2H), 4.53 (d, J=5.6 Hz, 2H), 4.40 (d, J=5.6 Hz, 2H), 4.02 (t, J=6.0 Hz, 2H), 3.95 (t, J=6.0 Hz, 2H), 2.30 (s, 3H), 2.15 (s, 3H); LC-MS: [M+H]⁺=287.1 m/z.

Step 2: (1-(2-aminoethyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanol and (1-(2-aminoethyl)-5-methyl-1H-1,2,3-triazol-4-yl)methanol: hydrazine hydrate (0.200 mL, 4.0 mmol) was added to a solution of N-(2-(4-(hydroxymethyl)-5-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide and N-(2-(5-(hydroxymethyl)-4-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide (0.575 g, 2.0 mmol) in ethanol (10 mL) and the mixture was heated to 60° C. for 2h. After completion, the mixture was filtered and filtrate was concentrated under reduced pressure to obtain the product as a mixture of isomers (0.305 g, crude) which was used as such in next step without purification. LC-MS: (M+H)⁺=157.2.

Step 3: N-(2-(4-(hydroxymethyl)-5-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide and N-(2-(5-(hydroxymethyl)-4-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide: HATU (0.685 g, 1.8 mmol) and DIPEA (0.58 g, 4.5 mmol) were added sequentially to a solution of 5-phenylthiazole-2-carboxylic acid (0.305 g, 1.5 mmol) in DMF (5 mL). After 10 min stirring, to the resulting reaction mixture was added (1-(2-aminoethyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanol and (1-(2-aminoethyl)-5-methyl-1H-1,2,3-triazol-4-yl)methanol (0.235 g, crude) and the reaction mixture was stirred at room temperature for 12 h. Reaction mixture was diluted with water (15 mL), extracted with dichloromethane (3×25 mL) and washed with brine (2×50 mL). Combined organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to obtain 0.9 g of crude material which was purified by prep HPLC to obtain target compounds. Yield: 25% over two steps.

N-(2-(5-(hydroxymethyl)-4-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide: 0.09 g; white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 8.98 (t, J=5.6 Hz, 1H), 8.41 (s, 1H), 7.77 (d, J=7.2 Hz, 2H), 7.50-7.40 (m, 3H), 5.32 (t, J=5.2 Hz, 1H), 4.54-4.51 (m, 4H), 3.73-3.69 (m, 2H), 2.20 (s, 3H); LC-MS: [M+H]⁺=344.0 m/z; HPLC purity 99.68% at 220 nm, 99.63% at 254 nm.

N-(2-(4-(hydroxymethyl)-5-methyl-1H-1,2,3-triazol-1-yl)ethyl)-5-phenylthiazole-2-carboxamide:0.085 g; white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.03 (t, J=5.6 Hz, 1H), 8.42 (s, 1H), 7.78 (d, J=7.2 Hz, 2H) 7.50-7.40 (m, 3H), 5.00 (t, J=5.6 Hz, 1H), 4.47-4.43 (m, 4H), 3.68-3.64 (m, 2H), 2.28 (s, 3H); LC-MS: [M+H]⁺=344.0 m/z; HPLC purity 99.75% at 220 nm, 99.62% at 254 nm.

Example 10 N-cis-3-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide

Step 1: ethyl 3-oxocyclobutane-1-carboxylate: triethyl orthoacetate (21.31 g, 0.131 mol) was added to a solution of 3-oxocyclobutane-1-carboxylic acid (5.0 g, 0.043 mol) in toluene (100 mL) and the reaction mixture was refluxed for 6 h. The reaction mixture was quenched with a IN HCl solution and the layers were separated off. The organic layer was washed with saturated NaHCO₃solution (2×50 mL), brine (2×50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the product (5.3 g, 85%) as a yellow liquid. ¹H NMR (400 MHz, CDCl₃): δ 4.23-4.17 (q, J=7.0 Hz, 2H), 3.44-3.37 (m, 2H), 3.32-3.16 (m, 3H), 1.30-1.26 (t, J=7.0 Hz, 3H).

Step 2: ethyl cis-3-hydroxycyclobutane-1-carboxylate: sodium borohydride (1.55 g, 0.041 mol) was added to an iced cold solution of ethyl 3-oxocyclobutane-1-carboxylate (5.3 g, 0.037 mol) in methanol (75 mL) and the reaction mixture was stirred for 1 h. The reaction mixture was quenched with acetone (10 mL) and volatiles were removed under reduced pressure. The crude reaction mixture was suspended in NaHCO₃solution (30 mL) and extracted with DCM (100 mL). The organic layer was washed with brine (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the product (3.2 g, 59%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 4.20-4.09 (overlapped q and m, 3H), 3.68 (d, J=2.3 Hz, 1H), 2.62-2.54 (m, 3H), 2.20-2.10 (m, 2H), 1.26-1.22 (t, J=7.0 Hz, 3H); LC-MS: [M+H]⁺ 145.1.

Step 3: ethyl cis-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate: Et₃N (8.96 mL, 0.0666 mol) was added to a solution of ethyl cis-3-hydroxycyclobutane-1-carboxylate (3.2 g, 0.0222 mol) in DCM (100 mL) followed by MsCl (3.03 g, 0.0266 mol) drop wise and the resulting reaction mixture was stirred at room temperature for 1 h. The reaction mixture was poured onto ice cold water (50 mL) and extracted with DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product (5.1 g) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 4.95-4.88 (m, 1H), 4.17-4.12 (q, J=7.1 Hz, 2H), 3.71 (d, 1H), 2.98 (s, 3H), 2.74-2.66 (m, 3H), 2.60-2.59 (m, 2H), 1.27-1.25 (t, J=7.1 Hz, 3H); LC-MS: [M+H]⁺ 223.0.

Step 4: ethyl trans-3-azidocyclobutane-1-carboxylate: a mixture of sodium azide (2.98 g, 0.044 mol) and ethyl cis-3-((methylsulfonyl)oxy)cyclobutane-1-carboxylate (5.1 g, 0.022 mol) in DMF (25 mL) was heated to 90° C. for 16 h. The reaction mixture was poured onto water (70 mL) and extracted with ethyl acetate (2×100 mL). Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain crude product which was chromatographed on 230-400 mesh silica gel using 10% EtOAc in hexane as eluent to afford the product (3.8 g, 100% over two steps) as colorless liquid. ¹H NMR (400 MHz, CDCl₃): δ 4.18-4.10 (m, 3H), 3.11-3.04 (m, 1H), 2.60-2.53 (m, 2H), 2.36-2.29 (m, 2H), 1.27-1.22 (t, J=7.1 Hz, 3H); LC-MS: [M+H]⁺ 171.1.

Step 5a/b: ethyl trans-3-aminocyclobutane-1-carboxylate hydrochloride: a mixture of ethyl trans-3-azidocyclobutane-1-carboxylate (3.8 g, 0.0221 mol) and 10% Pd/C (1.0 g) in ethanol (50 mL) was hydrogenated (50 psi) for 4 h at room temperature. The reaction mixture was filtered through a celite bed and the filtrate was concentrated under reduced pressure obtain the crude compound. The crude compound was treated with 4 M HCl in dioxane to afford HCl salt (3.8 g, 95%) as colorless viscous oil.

Step 6: ethyl trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carboxylate: Et₃N (0.15 mL, 0.0011 mol) and HATU (0.255 g, 0.00066 mol) were added sequentially to a suspension of ethyl trans-3-aminocyclobutane-1-carboxylate hydrochloride (0.100 g, 0.00055 mol) and 5-phenylthiazole-2-carboxylic acid (0.125 g, 0.000612 mol) in THF (10 mL) and the reaction mixture was stirred for 12 h at room temperature. Volatiles were removed under reduced pressure and the crude reaction mixture was diluted with water (10 mL). The aq. phase was extracted with ethyl acetate (2×25 mL). Combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude compound which was further purified by (230-400 mesh) silica gel column chromatography using 25% EtOAc in hexane to afford the product (0.130 g, 70%) as off white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.97 (s, 1H), 7.60-7.57 (m, 2H), 7.45-7.36 (m, 4H), 4.76-4.74 (m, 1H), 4.18 (q, J=7.0 Hz, 2H), 3.11-3.10 (m, 1H), 2.78-2.72 (m, 2H), 2.43-2.35 (m, 2H), 1.28 (t, J=7.0 Hz, 3H); LC-MS: [M+H]⁺ 330.9 m/z.

Step 7: trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carboxylic acid: lithium hydroxide monohydrate (0.153 g, 0.0036 mol) was added to a solution of ethyl trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carboxylate (1.1 g, 0.0033 mol) in THF:water (45 mL, 2:1) and the resultant reaction mixture was stirred at room temperature for 2 h. The reaction mixture was evaporated under vacuum and partitioned between ethyl acetate (30 mL) and water (10 mL). Separated aqueous layer was acidified with saturated citric acid solution (15 mL), precipitate thus formed was filtered, washed with water and dried to afford the product (0.315 g, 98%) as white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 12.21 (s, 1H), 9.25 (d, J=8.1 Hz, 1H), 8.42 (s, 1H), 7.77 (d, J=7.3 Hz, 2H), 7.50-7.40 (m, 3H), 4.60-4.54 (m, 1H), 2.96-2.91 (m, 1H), 2.50-2.39 (m, 4H); LC-MS: [M+H]⁺ 303.1 m/z.

Step 8: tert-butyl 2-trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carbonyl)hydrazine-1-carboxylate: HATU (1.49 g, 0.0039 mol) was added to a solution of trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carboxylic acid (0.990 g, 0.0032 mol) in THF (90 mL) and stirred at room temperature for 15 min. To the resulting reaction mixture was added N-Boc-hydrazine (0.518 g, 0.0039 mol) followed by Et₃N (0.88 mL, 0.00654 mol) and stirring continued for 2 h at room temperature. The reaction mixture was poured onto water (80 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude compound. The crude compound was further purified by silica gel (230-400 mesh) column chromatography using EtOAc as eluent to afford the product (1.8 g, crude) as white solid with polar impurity. LC-MS: [M+H]⁺ 417.2 m/z.

Step 9: N-trans-3-(hydrazinecarbonyl)cyclobutyl)-5-phenylthiazole-2-carboxamide hydrochloride: 4 M HCl in dioxane solution (12 mL) was added to a solution of tert-butyl 2-trans-3-(5-phenylthiazole-2-carboxamido)cyclobutane-1-carbonyl)hydrazine-1-carboxylate (0.800 g, 0.00192 mol) in 1,4-dioxane (5 mL) and the reaction mixture was stirred at room temperature for 6 h. Volatiles were removed under reduced pressure and the reaction mixture was diluted with diethyl ether (100 mL) to afford the product (0.650 g, crude) as off white solid. LC-MS: [M+H]⁺ 317.2 m/z.

Step 10: N-trans-3-(2-(2-((tert-butyldimethylsilyl)oxy)acetyl)hydrazine-1-carbonyl)cyclobutyl)-5-phenylthiazole-2-carboxamide: HATU (0.256 g, 0.00067 mol) was added to a solution of N-trans-3-(hydrazinecarbonyl)cyclobutyl)-5-phenylthiazole-2-carboxamide hydrochloride (0.161 g, 0.00085 mol) in THF (20 mL) and stirred at room temperature for 15 minutes. Then added 2-((tert-butyldimethylsilyl)oxy)acetic acid (0.200 g, 0.00056 mol) and Et₃N (0.301 mL, 0.00224 mol) were added and the mixture was stirred at room temperature for 6 h. The reaction mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure obtained crude product. The crude compound was purified by column chromatography on 230-400 mesh silica gel using 90% EtOAc in hexane afforded the product (0.103 g, crude) as off white solid. LC-MS: [M+H]⁺ 489.1 m/z.

Step 11: N-trans-3-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide: Iodine (0.206 g, 0.0008 mol) was added to a solution of PPh₃(0.212 g, 0.00081 mol) in DCM (30 mL) at room temperature and the reaction mixture was stirred for 15 min. The resulting reaction mixture was cooled in an ice bath and to the reaction mixture was triethyl amine (0.27 mL, 0.00204 mol) followed by N-trans-3-(2-(2-((tert-butyldimethylsilyl)oxy)acetyl)hydrazine-1-carbonyl)cyclobutyl)-5-phenylthiazole-2-carboxamide (0.200 g, 0.00040 mol). The reaction mixture was stirred at room temperature for 3 h. Volatiles were removed under reduced pressure; the crude compound was diluted with ethyl acetate and filtered. The filtrate was concentrated under reduced pressure to afford crude product. The crude compound was purified by (230-400 mesh) silica gel column chromatography using 60% EtOAc in hexane as eluent to obtain the product (0.110 g, 21% over four steps) as off white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.41 (d, J=8.0 Hz, 1H), 8.44 (s, 1H), 7.79-7.77 (m, 2H), 7.50-7.47 (m, 2H), 7.43-7.41 (m, 1H), 4.87 (s, 2H), 4.73-4.67 (m, 1H), 3.73-3.69 (m, 1H), 2.80-2.72 (m, 2H), 2.60-2.54 (m, 2H), 0.88 (s, 9H), 0.10 (s, 6H); LC-MS: [M+H]⁺ 471.3 m/z.

Step 12: N-trans-3-(5-(hydroxymethyl)-1,3,4-oxadiazol-2-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide: tetra butyl ammonium fluoride (0.46 mL, 0.00046 mol, 1 M in THF solution) was added to a cold solution of N-trans-3-(5-(((tert-butyldimethylsilyl)oxy)methyl)-1,3,4-oxadiazol-2-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide (0.110 g, 0.00023 mol) in THF (5 mL) and the reaction mixture was stirred for 1 h. Volatiles were removed under reduced pressure; the crude reaction mixture was suspended in water (10 mL) and extracted with ethyl acetate (15 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure to afford the product (0.070 g, 84%) as off white solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.41 (d, J=8 Hz, 1H), 8.45 (s, 1H), 7.80-7.77 (m, 2H), 7.50-7.42 (m, 3H), 5.89-5.86 (m, 1H), 4.72-4.67 (m, 1H), 4.63 (d, J=4 Hz, 2H), 3.72-3.67 (m, 1H), 2.79-2.71 (m, 2H), 2.61-2.56 (m, 2H); LC-MS: [M+H]⁺ 356.8 m/z; HPLC purity: 99.39% at 220 nm and 98.86% at 254 nm.

Example 11 N-trans-3-(5-((R)-1-hydroxyethyl)-1,3,4-oxadiazol-2-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide

Step 1a: methyl (2R)-2-[(tert-butyldimethylsilyl)oxy]propanoate: into a 250-mL round-bottom flask, was placed a solution of methyl (2R)-2-hydroxypropanoate (5 g, 48.03 mmol, 1.00 equiv) and Imidazole (6.5 g, 95.59 mmol, 2.00 equiv) in dichloromethane (100 mL). This was followed by the addition of a solution of tert-butyl(chloro)dimethylsilane (8.7 g, 57.72 mmol, 1.20 equiv) in dichloromethane (50 mL) dropwise with stirring at 0° C. The resulting solution was stirred for 2 hours at room temperature. The reaction was then quenched by the addition of 100 mL of water/ice. The resulting solution was extracted with dichloromethane (3×100 mL) and the organic layers combined. The resulting mixture was washed with brine (3×50 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 7 g (67%) of methyl (2R)-2-[(tert-butyldimethylsilyl)oxy]propanoate as colorless oil.

Step 1b: (2R)-2-[(tert-butyldimethylsilyl)oxy]propanehydrazide: into a 250-mL round-bottom flask, was placed a solution of methyl (2R)-2-[(tert-butyldimethylsilyl)oxy]propanoate (7 g, 32.06 mmol, 1.00 equiv) in ethanol (100 mL). To the solution was added hydrazine (10 g, 159.81 mmol, 5.00 equiv, 80%). The resulting solution was stirred for 15 hours at 90° C. in an oil bath. The resulting solution was quenched by the addition of water/ice. The resulting solution was extracted with ethyl acetate (3×100 mL) and the organic layers combined. The resulting mixture was washed with brine (2×100 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 6.5 g (93%) of (2R)-2-[(tert-butyldimethylsilyl)oxy]propanehydrazide as colorless oil. LC-MS (ES, m/z): [M+1]⁺=219.

Step 1: methyl (trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylate: into a 250-mL round-bottom flask, under nitrogen, was placed a solution of methyl 3-cis-hydroxycyclobutane-1-carboxylate (8 g, 61.47 mmol, 1.00 equiv), 2,3-dihydro-1H-isoindole-1,3-dione (18.1 g, 123.02 mmol, 2.00 equiv) and triphenylphosphine (32.3 g, 123.15 mmol, 2.00 equiv) in THF (100 mL). This was followed by the addition of DIAD (24.9 g, 123.14 mmol, 2.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 2.5 hours at room temperature. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:5). The crude product was re-crystallized from petroleum ether/ethyl acetate in the ratio of 10:1. This resulted in 7.2 g (45%) of methyl trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylate as a white solid. LC-MS (ES, m/z): [M+1]⁺=260. ¹H NMR (400 MHz, CDCl₃): δ 7.85-7.82 (m, 2H), 7.74-7.71 (m, 2H), 5.08-5.04 (m, 1H), 3.75 (s, 3H), 3.34-3.32 (m, 1H), 3.20-3.12 (m, 2H), 2.66-2.60 (m, 2H).

Step 2: trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylic acid: into a 100-mL round-bottom flask, was placed a solution of methyl trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylate (7.2 g, 27.77 mmol, 1.00 equiv) in 1,4-dioxane (100 mL). To the solution was added 5M hydrogen chloride aqueous (10 mL). The resulting solution was stirred for 4 hours at 80° C. in an oil bath. The resulting mixture was concentrated under vacuum. This resulted in 6.2 g (91%) of trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylic acid as a white solid. LC-MS (ES, m/z): [M−1]⁻=244.

Step 3: (2R)-2-[(tert-butyldimethylsilyl)oxy]-N-[trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutyl]carbonyl]propanehydrazide: into a 250-mL round-bottom flask, was placed a solution of trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutane-1-carboxylic acid (6.2 g, 25.28 mmol, 1.00 equiv), (2R)-2-[(tert-butyldimethylsilyl)oxy]propanehydrazide (6.61 g, 30.27 mmol, 1.20 equiv) and HATU (14.4 g, 37.89 mmol, 1.50 equiv) in THF (100 mL). This was followed by the addition of DIEA (9.81 g, 75.91 mmol, 3.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 1 hour at room temperature. The reaction was then quenched by the addition of 100 mL of water/ice. The resulting solution was extracted with ethyl acetate (3×50 mL) and the organic layers combined. The resulting mixture was washed with brine (2×50 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:4). This resulted in 7 g (62%) of (2R)-2-[(tert-butyldimethylsilyl)oxy]-N-[trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutyl]carbonyl]propanehydrazide as colorless oil. LC-MS (ES, m/z): [M+1]⁺=446.

Step 4: 2-[trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-2,3-dihydro-1H-isoindole-1,3-dione: into a 250-mL round-bottom flask, was placed a solution of (2R)-2-[(tert-butyldimethylsilyl)oxy]-N-[[trans-3-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)cyclobutyl]carbonyl]propanehydrazide (6.95 g, 15.60 mmol, 1.00 equiv) and TEA (7.89 g, 77.97 mmol, 5.00 equiv) in dichloromethane (100 mL). This was followed by the addition of a solution of 4-methylbenzene-1-sulfonyl chloride (8.92 g, 46.79 mmol, 3.00 equiv) in dichloromethane (50 mL) dropwise with stirring at 0° C. The resulting solution was stirred for 15 hours at room temperature. The reaction was then quenched by the addition of 100 mL of water/ice. The resulting solution was extracted with dichloromethane (2×50 mL) and the organic layers combined. The resulting mixture was washed with brine (2×50 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18; mobile phase, H₂O/CH₃CN=100:1 increasing to H₂O/CH₃CN=1:100 within 30 min; Detector, UV 254 nm. This resulted in 3.28 g (49%) of 2-[trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-2,3-dihydro-1H-isoindole-1,3-dione as colorless oil. LC-MS (ES, m/z): [M+1]⁺=428. ¹H NMR (400 MHz, CDCl₃): δ 7.72-7.70 (m, 2H), 7.60-7.58 (m, 2H), 5.04-4.96 (m, 2H), 3.83-3.78 (m, 1H), 3.26-3.24 (m, 2H), 2.67-2.62 (m, 2H), 1.49-1.48 (d, J=6.8 Hz, 3H), 0.76 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H).

Step 5: trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutan-1-amine: into a 250-mL round-bottom flask, was placed a solution of 2-[trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-2,3-dihydro-1H-isoindole-1,3-dione (1.18 g, 2.76 mmol, 1.00 equiv) in ethanol (100 mL). To the solution was added hydrazine hydrate (3.45 g, 55.13 mmol, 20.00 equiv, 80%). The resulting solution was stirred for 3 hours at room temperature. The solids were filtered out. The resulting mixture was concentrated under vacuum. This resulted in 760 mg (crude) of trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutan-1-amine as colorless oil. LC-MS (ES, m/z): [M+1]⁺=298.

Step 6: N-[trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-1,3-thiazole-2-carboxamide: into a 100-mL round-bottom flask, was placed a solution of lithio 5-phenyl-1,3-thiazole-2-carboxylate (332 mg, 1.57 mmol, 1.20 equiv), (1r,3r)-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutan-1-amine (390 mg, 1.31 mmol, 1.00 equiv) and HATU (750 mg, 1.97 mmol, 1.50 equiv) in THF (50 mL). This was followed by the addition of DIEA (508 mg, 3.93 mmol, 3.00 equiv) dropwise with stirring at 0° C. The resulting solution was stirred for 1 hour at room temperature. The resulting solution was diluted with 50 mL of water/ice. The resulting solution was extracted with ethyl acetate (3×50 mL) and the organic layers combined. The resulting mixture was washed with brine (2×30 mL), dried and concentrated under vacuum. This resulted in 300 mg (47%) of 5-phenyl-N-[trans-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-1,3-thiazole-2-carboxamide as a light yellow crude solid. LC-MS (ES, m/z): [M+1]⁺=485.

Step 7: 5-phenyl-N-[trans-3-[5-[(1R)-1-hydroxyethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-1,3-thiazole-2-carboxamide: into a 50-mL round-bottom flask, was placed a solution of 5-phenyl-N-[(1r,3r)-3-[5-[(1R)-1-[(tert-butyldimethylsilyl)oxy]ethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-1,3-thiazole-2-carboxamide (300 mg, 0.62 mmol, 1.00 equiv) in THF (2 mL). To the solution was added TBAF (1 mol/L in tetrahydrofuran, 1 mL). The resulting solution was stirred for 3 hours at room temperature. The resulting solution was diluted with 20 mL of water. The resulting solution was extracted with ethyl acetate (3×30 mL) and the organic layers combined. The resulting mixture was washed with brine (2×10 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (20:1). The crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18; mobile phase, H₂O/CH₃CN=100:1 increasing to H₂O/CH₃CN=1:100 within 30 min; Detector, UV 254 nm. This resulted in 115.6 mg (50%) of 5-phenyl-N-[trans-3-[5-[(1R)-1-hydroxyethyl]-1,3,4-oxadiazol-2-yl]cyclobutyl]-1,3-thiazole-2-carboxamide as a white solid. LC-MS (ES, m/z): [M+1]⁺=371. ¹H NMR (400 MHz, DMSO-d₆): δ 9.42-9.40 (d, J=8.0 Hz, 1H), 8.43 (s, 1H), 7.80-7.78 (m, 2H), 7.51-7.42 (m, 3H), 5.96-5.94 (d, J=6.0 Hz, 1H), 4.95-4.90 (m, 1H), 4.75-4.65 (m, 1H), 3.73-3.67 (m, 1H), 2.80-2.72 (m, 2H), 2.63-2.57 (m, 2H), 1.50-1.48 (d, J=6.4 Hz, 3H).

Example 12 N-trans-3-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)cyclobutyl)-5-phenylthiazole-2-carboxamide

Step 1: tert-butyl (3-oxocyclobutyl)carbamate: DPPA (4.0 g, 1.1 eq.) is added dropwise to a cold (−5˜5° C.) solution of 3-oxocyclobutanecarboxylic acid (1.5 g, 1.0 eq.) and TEA (1.5 g, 1.1 eq.) in toluene (30 mL), and the mixture is stirred at −5˜0° C. for 16 h. The reaction mixture is washed with NaHCO₃(2*9 mL), water (1*9 mL) and NaCl aq. (1*4.5 mL) at 0˜10° C. The organic phase is dried over Na₂SO₄, filtered and t-BuOH (7.5 mL) is added to the filtrate. The reaction mixture is heated at 90˜100° C. for 16 h. The mixture is concentrated under vacuum at 60˜70° C. and then suspended in TBME (4.5 mL), filtered and the solid is dried over air to give 1.15 g (purity: 98.5%, yield: 47.2%) of product as a white solid.

Step 2: tert-butyl (cis-3-hydroxycyclobutyl)carbamate: a solution of tert-butyl (3-oxocyclobutyl)carbamate (200 mg, 1.0 eq.) in THF (1 mL) is added dropwise to a cold (below −70° C.) solution of NaBH₄(20.4 mg, 0.5 eq.) in a solution of THF (1.8 mL) and water (2 mL), maintaining the temperature at −80˜−70° C. (ca. for 2 h for completion of addition). The mixture is stirred at −60˜−50° C. for 3 h, water (2 mL) is added to the reaction mixture and allowed to reach up to 15° C. The reaction mixture is then extracted with ethyl acetate (2 mL, 2*1 mL) and the combined organic layers are washed with brine (1 mL). The organic layer is concentrated under vacuum at 35˜40° C., the solid is dissolved in toluene (1 mL, 80˜90° C.) and then is gradually cooled to 25-30° C. for 2.5 h. The mixture is stirred for 2 h at 25-30° C., filtered, and the solid is dried in the air to give the product (177 mg with ratio of cis:trans (96.4:3.6), yield: 87.6%) as an off-white solid.

Step 3: tert-butyl (trans-3-azidocyclobutyl)carbamate: a solution of PPh₃(315 mg) and DIAD (243 mg) in THF (3 mL) is stirred for 20 min at 0-10° C. Then a solution of tert-butyl (cis-3-hydroxycyclobutyl)carbamate (150 mg, 1.0 eq.) and DPPA (265 mg, 1.2 eq.) in THF (1 ml) is added dropwise, the mixture is then warmed to 25-30° C. and it is stirred for 2 h. Brine (3 mL) is added to the reaction mixture, extracted with ethyl acetate (3 mL) and is then concentrated under vacuum to give the crude oil. The mixture is purified by SiO₂column chromatography and is eluted with ethyl acetate/petroleum ether (0%˜10%) gradually. The product is suspended in n-heptane (0.3 mL) and stirred for 0.5 h at 20˜25° C., the mixture is filtered and the solid is dried in air to give the product in 85% yield and ratio of cis/trans=4:96 checked by ¹H NMR.

Step 4: tert-butyl (trans-3-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)cyclobutyl)carbamate: a solution of tert-butyl (trans-3-azidocyclobutyl)carbamate (246 mg, 1.0 eq.) and prop-2-yn-1-ol (326 mg, 5.0 eq.) in DMF (1.2 mL) is heated at 90˜95° C. for 20 h. The mixture is concentrated under vacuum at 65° C. to give ˜1:1 mixture of 4 and 5 regioisomers (353 mg). The mixture is purified by SFC to give tert-butyl (trans-3-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)cyclobutyl)carbamate (101 mg, P: 99.9% (205 nm), Y: 32%) as a solid.

Step 5: (1-(trans-3-aminocyclobutyl)-1H-1,2,3-triazol-5-yl)methanol hydrochloride: tert-butyl (trans-3-(5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)cyclobutyl)carbamate (101 mg, 1.0 eq.) is added slowly (5 portions) to a solution of HCl/dioxane (3.5 mol/L, 2 mL) at 20˜30° C., and then is stirred for 18 h at 20˜30° C. The reaction mixture is concentrated under vacuum at 55° C. to give the product (93.4 mg, assay 67% based on free base, Y: 100%) as a solid.

Step 6: of 5-phenyl-N-trans-3-[5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]cyclobutyl]-1,3-thiazole-2-carboxamide as a white solid: DIEA (386 mg, 2.99 mmol, 3.00 equiv) was added dropwise to a cold solution 0° C. of lithio 5-phenyl-1,3-thiazole-2-carboxylate (180 mg, 0.85 mmol, 1.00 equiv), [1-[trans-3-aminocyclobutyl]-1H-1,2,3-triazol-5-yl]methanol hydrochloride (204 mg, 1.00 mmol, 1.00 equiv) and HATU (567 mg, 1.49 mmol, 1.50 equiv) in DMF (5 mL). The resulting solution was stirred for 1 hour at room temperature. The reaction was then quenched by the addition of 50 mL of water/ice. The resulting solution was extracted with ethyl acetate (3×80 mL) and the organic layers combined. The resulting mixture was washed with brine (3×30 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The crude mixture was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18; mobile phase, H₂O/CH₃CN=100:1 increasing to H₂O/CH₃CN=1:100 within 35 min; Detector, UV 254 nm. This resulted in 115 mg (38%) of 5-phenyl-N-trans-3-[5-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]cyclobutyl]-1,3-thiazole-2-carboxamide as a white solid. LC-MS (ES, m/z): [M+1]⁺=356. ¹H NMR (400 MHz, DMSO-d₆): δ 9.47-9.45 (d, J=8.0 Hz, 1H), 8.45 (s, 1H), 7.81-7.79 (d, J=7.6 Hz, 2H), 7.65 (s, 1H), 7.52-7.42 (m, 3H), 5.43 (s, 1H), 5.26-5.19 (m, 1H), 4.85-4.76 (m, 1H), 4.56 (s, 2H), 2.90-2.81 (m, 4H).

Example 13 CFTR Activity Assays

i. Ussing Measurements

As discussed above, Ussing measurements are used to measure CFTR activity. In this method, primary lung epithelial cells (hBEs) homozygous for the Cystic Fibrosis-causing ΔF508 mutation are differentiated for a minimum of 4 weeks in an air-liquid interface on SnapWell filter plates prior to the Ussing measurements. Cells are apically mucus-washed for 30 minutes prior to treatment with compounds. The basolateral media is removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO stocks. Treated cells are incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the cells on filters are transferred to the Ussing chamber and equilibrated for 30 minutes. The short-circuit current is measured in voltage clamp-mode (V_hold=0 mV), and the entire assay is conducted at a temperature of 36° C. −36.5° C. Once the voltages stabilized, the chambers are clamped, and data is recorded by pulse readings every 5 seconds. Following baseline current stabilization, the following additions can be applied and the changes in current and resistance of the cells can be monitored:

- 1. Benzamil to the apical chamber to inhibit ENaC sodium channel.
- 2. Forskolin to both chambers to activate ΔF508-CFTR by phosphorylation.
- 3. Genistein to both chambers to potentiate ΔF508-CFTR channel opening.
- 4. CFTRinh-172 to the apical chamber to inhibit ΔF508-CFTR Cl-conductance.

The inhibitable current (that current that is blocked by CFTRinh-172) is measured as the specific activity of the ΔF508-CFTR channel, and increases in response to compound in this activity over that observed in vehicle-treated samples are identified as the correction of ΔF508-CFTR function imparted by the compound tested.

ii. hBE Equivalent Current (Ieq) Assay

Primary lung epithelial cells homozygous for the Cystic Fibrosis-causing ΔF508 mutation were differentiated for a minimum of 4 weeks in an air-liquid interface on Costar 24 well HTS filter plates prior to the equivalent current (Ieq) measurements. Cells were apically mucus-washed for 30 minutes 24h prior to treatment with compounds. The basolateral media was removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO stocks. Treated cells were incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the media was changed to the Ieq experimental solution for 30 minutes before the experiment and plates are maintained in a CO₂-free incubator during this period. The plates containing the cells were then placed in pre-warmed heating blocks at 36° C.±0.5 for 15 minutes before measurements are taken. The transepithelial voltage (V_T) and conductance (G_T) were measured using a custom 24 channel current clamp (TECC-24) with 24 well electrode manifold. The Ieq assay measurements were made following additions with standardized time periods:

- 1. The baseline V_Tand G_Tvalues were measured for approximately 20 minutes.
- 2. Benzamil was added to block ENaC for 15 minutes.
- 3. Forskolin plus VX-770 were added to maximally activate ΔF508-CFTR for 27 minutes.
- 4. Bumetanide was added to inhibit the NaK₂Cl cotransporter and shut-off secretion of chloride.

The activity data captured was the area under the curve (AUC) for the traces of the equivalent chloride current. The AUC was collected from the time of the forskolin/VX-770 addition until the inhibition by bumetanide addition. Correction in response to compound treatment was scored as the increase in the AUC for compound-treated samples over that of vehicle-treated samples. The results are shown below in Table 2. (** indicates activity ≥200% of VX-809 (1 uM) with compound at 10 uM and VX-809 at 1 uM; * indicates activity 100-200% of VX-809 (1 uM) with compound at 10 uM and VX-809 at 1 uM. ^##indicates activity ≥200% of VX-809 (3 uM) with compound at 10 uM and VX-809 at 3 uM; ^#indicates activity 100-200% of VX-809 (3 uM) with compound at 10 uM and VX-809 at 3 uM

TABLE 2

#	Structure	leq	Ussing

1	**

2	**

3	**

4	**

5	*

6	**	##

7	**	##

8	**	##

9	**

10		##

11		##

12		##

Example 14

i. Ussing Measurements

As discussed above, Ussing measurements can be used to measure CFTR activity. In this method, primary lung epithelial cells (hBEs) with a Cystic fibrosis causing class I mutation are differentiated for a minimum of 4 weeks in an air-liquid interface on SnapWell™ filter plates prior to the Ussing measurements. Cells are apically mucus-washed for 30 minutes prior to treatment with compounds. The basolateral media is removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO or aqueous stocks. Treated cells are incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the cells on filters are transferred to the Ussing chamber and equilibrated for 30 minutes. The short-circuit current is measured in voltage clamp-mode (V_hold=0 mV), and the entire assay is conducted at a temperature of 36° C.-36.5° C. Once the voltages stabilize, the chambers are clamped, and data are recorded by pulse readings every 5 seconds. Following baseline current stabilization, the following additions are applied and the changes in current and resistance of the cells are monitored:

- 1. Benzamil to the apical chamber to inhibit ENaC sodium channel.
- 2. Forskolin to both chambers to activate ΔF508-CFTR by phosphorylation.
- 3. Ivacaftor or Genistein to the apical chamber to potentiate ΔF508-CFTR channel opening.
- 4. CFTRinh-172 to the apical chamber to inhibit ΔF508-CFTR Cl-conductance.

The forskolin-sensitive current and inhibitable current (that potentiated current that is blocked by CFTRinh-172) are measured as the specific activity of the ΔF508-CFTR channel, and increase in response to compound in this activity over that observed in vehicle-treated samples are identified as the correction of ΔF508-CFTR function imparted by the compound tested.

Example 15

i. Ussing Measurements

As discussed above, Ussing measurements can be used to measure CFTR activity. In this method, primary lung epithelial cells (hBEs) with a Cystic Fibrosis-causing class III mutation are differentiated for a minimum of 4 weeks in an air-liquid interface on SnapWell™ filter plates prior to the Ussing measurements. Cells are apically mucus-washed for 30 minutes prior to treatment with compounds. The basolateral media is removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO stocks. Treated cells are incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the cells on filters are transferred to the Ussing chamber and equilibrated for 30 minutes. The short-circuit current is measured in voltage clamp-mode (V_hold=0 mV), and the entire assay is conducted at a temperature of 36° C.-36.5° C. Once the voltages stabilize, the chambers are clamped, and data is recorded by pulse readings every 5 seconds. Following baseline current stabilization, the following additions are applied and the changes in current and resistance of the cells is monitored:

- 1. Benzamil to the apical chamber to inhibit ENaC sodium channel.
- 2. Forskolin to both chambers to activate ΔF508-CFTR by phosphorylation.
- 3. VX-770 or Genistein to the apical chamber to potentiate ΔF508-CFTR channel opening.
- 4. CFTRinh-172 to the apical chamber to inhibit ΔF508-CFTR Cl-conductance.

Example 16

i. Ussing Measurements

As discussed above, Ussing measurements can be used to measure CFTR activity. In this method, primary lung epithelial cells (hBEs) with a Cystic Fibrosis-causing class V mutation are differentiated for a minimum of 4 weeks in an air-liquid interface on SnapWell™ filter plates prior to the Ussing measurements. Cells are apically mucus-washed for 30 minutes prior to treatment with compounds. The basolateral media is removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO stocks. Treated cells are incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the cells on filters are transferred to the Ussing chamber and equilibrated for 30 minutes. The short-circuit current is measured in voltage clamp-mode (V_hold=0 mV), and the entire assay is conducted at a temperature of 36° C.-36.5° C. Once the voltages stabilize, the chambers are clamped, and data is recorded by pulse readings every 5 seconds. Following baseline current stabilization, the following additions are applied and the changes in current and resistance of the cells is monitored:

- 1. Benzamil to the apical chamber to inhibit ENaC sodium channel.
- 2. Forskolin to both chambers to activate ΔF508-CFTR by phosphorylation.
- 3. VX-770 or Genistiein to the apical chamber to potentiate ΔF508-CFTR channel opening.
- 4. CFTRinh-172 to the apical chamber to inhibit ΔF508-CFTR Cl-conductance.

The forskolin-sensitive current and inhibitable current (that potentiated current that is blocked by CFTRinh-172) are measured as the specific activity of the ΔF508-CFTR channel, and increases in response to compound in this activity over that observed in vehicle-treated samples are identified as the correction of ΔF508-CFTR function imparted by the compound tested.

ii. hBE Equivalent Current (Ieq) Assay

Primary lung epithelial cells homozygous for the Cystic Fibrosis-causing ΔF508 mutation are differentiated for a minimum of 4 weeks in an air-liquid interface on Costar 24 well HTS filter plates prior to the equivalent current (Ieq) measurements. Cells are apically mucus-washed for 30 minutes 24h prior to treatment with compounds. The basolateral media is removed and replaced with media containing the compound of interest diluted to its final concentration from DMSO stocks. Treated cells are incubated at 37° C. and 5% CO₂for 24 hours. At the end of the treatment period, the media is changed to the Ieq experimental solution for 30 minutes before the experiment and plates are maintained in a CO₂-free incubator during this period. The plates containing the cells are then placed in pre-warmed heating blocks at 36° C.±0.5 for 15 minutes before measurements are taken. The transepithelial voltage (V_T) and conductance (G_T) are measured using a custom 24 channel current clamp (TECC-24) with 24 well electrode manifold. The Ieq assay measurements are made following additions with standardized time periods:

- 1. The baseline V_Tand G_Tvalues are measured for approximately 20 minutes.
- 2. Benzamil is added to block ENaC for 15 minutes.
- 3. Forskolin plus VX-770 (ivacaftor) are added to maximally activate ΔF508-CFTR for 27 minutes.
- 4. Bumetanide is added to inhibit the NaK₂Cl cotransporter and shut-off secretion of chloride.

The activity data captured is the area under the curve (AUC) for the traces of the equivalent chloride current. The AUC is collected from the time of the forskolin/VX-770 addition until the inhibition by bumetanide addition. Correction in response to compound treatment is scored as the increase in the AUC for compound-treated samples over that of vehicle-treated samples.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

Claims

What is claimed is:

1. A compound represented by formula III or IV:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein;

X₃is selected from the group consisting of O, S, and NR_hh;

pp is 1, 2, or 3;

R₁₁is independently selected for each occurrence from the group consisting of hydrogen, halogen, and C_1-4alkyl (optionally substituted by one, two or three halogens);

L₁is C_3-6cycloalkylene, wherein L₁may be optionally substituted by one, two or three substituents selected from the group consisting of halogen, hydroxyl, and C_1-3alkyl (optionally substituted by one, two or three substituents each selected independently from R_ff);

R₄₄is a 5-6 membered monocyclic heteroaryl having one, two or three heteroatoms each selected from O, N, and S; wherein heteroaryl may be optionally substituted by one or two substituents each selected independently from R_gg;

R_ffis selected for each occurrence from the group consisting of halogen, hydroxyl, C_1-4alkyl, C_1-4alkoxy, C_2-4alkenyl, C_3-6cycloalkyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)_w—C_1-3alkyl, where w is 0, 1, or 2, wherein C_1-4alkyl, C_1-4alkoxy, C_2-4alkenyl and C_3-6cycloalkyl may be optionally substituted by one, two or three substituents each independently selected from the group consisting of halogen, hydroxyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_W—NR′R″, and —S(O)_W—C_1-3alkyl;

R_ggis selected for each occurrence from the group consisting of halogen, hydroxyl, C_1-6alkyl, C_1-6alkoxy, C_2-6alkenyl, C_3-6cycloalkyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_w—NR′R″, and —S(O)_w—C_1-3alkyl, where w is 0, 1, or 2, wherein C_1-6alkyl, C_1-6alkoxy C_2-6alkenyl and C_3-6cycloalkyl may each be optionally substituted by one, two or three substituents each independently selected from the group consisting of halogen, C_1-6alkyl, C_1-6alkoxy, hydroxyl, C(O)OH, —C(O)OC_1-6alkyl, —O—C_3-6cycloalkyl, —O-heterocycle, —O-heteroaryl, —O-phenyl, —NR′R″, —NR′—S(O)_w—C_1-3alkyl, S(O)_W—NR′R″, and —S(O)w-C_1-3alkyl;

w is 0, 1 or 2; and

2. The compound of claim 1, represented by:

wherein qq is 0 or 1.

3. The compound of claim 1, represented by:

4. The compound of claim 1, wherein R₄₄is selected from the group consisting of:

wherein X₂is selected from the group consisting of O, S and NR_hh; R″ is H or C_1-4alkyl, and each R₆₆, R₇₇, and R₈₈is independently selected from H and R_gg.

5. The compound of claim 4, where each R₆₆, R₇₇, and R₈₈is independently selected from the group consisting of hydrogen, halogen, hydroxyl, C_1-6alkyl, C_3-6cycloalkyl, and heterocycle, wherein C_1-6alkyl, C_3-6cycloalkyl, and heterocycle are optionally substituted by one, two or three substituents each independently selected from the group consisting of hydroxyl, C_1-6alkyl, C_1-6alkoxy (optionally substituted by C_3-6cycloalkyl, heterocycle, —C_1-2alkyl-heterocycle and C_1-2alkyl-C_3-6cycloalkyl) , —S(O)_w—C _1-3alkyl (w is 0,1, or 2) and —NR′S(O)₂C_1-6alkyl; and

R′ is independently selected for each occurrence from H and C_1-4alkyl.

6. The compound of claim 1, wherein pp is 1 or 2, and R₁₁is selected from H, F, or methyl.

7. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier or excipient.